U.S. patent application number 13/196436 was filed with the patent office on 2012-04-19 for compositions for bacterial mediated gene silencing and methods of using same.
Invention is credited to Johannes Heinrich Fruehauf, Chiang Li, Shuanglin Xiang.
Application Number | 20120093773 13/196436 |
Document ID | / |
Family ID | 36588572 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120093773 |
Kind Code |
A1 |
Li; Chiang ; et al. |
April 19, 2012 |
Compositions for Bacterial Mediated Gene Silencing and Methods of
Using Same
Abstract
Methods are described for the delivery of one or more small
interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium.
Methods are also described for using this bacterium to regulate
gene expression in eukaryotic cells using RNA interference, and
methods for treating cancer of cell proliferative disorders. The
bacterium includes one or more siRNAs or one or more DNA molecules
encoding one or more siRNAs. Vectors are also described for use
with the bacteria of the invention for causing RNA interference in
eukaryotic cells.
Inventors: |
Li; Chiang; (Cambridge,
MA) ; Fruehauf; Johannes Heinrich; (Newton, MA)
; Xiang; Shuanglin; (Boston, MA) |
Family ID: |
36588572 |
Appl. No.: |
13/196436 |
Filed: |
August 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11793429 |
Nov 20, 2007 |
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PCT/US05/45513 |
Dec 16, 2005 |
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13196436 |
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60637277 |
Dec 17, 2004 |
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60651238 |
Feb 8, 2005 |
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Current U.S.
Class: |
424/93.2 ;
435/252.3; 435/252.33; 435/320.1; 435/350; 435/351; 435/363;
435/366; 435/375 |
Current CPC
Class: |
C12N 2310/111 20130101;
C12N 2320/32 20130101; C12N 15/113 20130101; C12N 15/111 20130101;
C12N 15/1138 20130101; C12N 15/8218 20130101; A61P 35/00 20180101;
C12N 15/1135 20130101; C12N 2310/531 20130101; C12N 2310/14
20130101; A61K 35/74 20130101; C12N 15/1137 20130101 |
Class at
Publication: |
424/93.2 ;
435/375; 435/252.3; 435/320.1; 435/363; 435/366; 435/350; 435/351;
435/252.33 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61P 35/00 20060101 A61P035/00; C12N 15/63 20060101
C12N015/63; C12N 5/071 20100101 C12N005/071; C12N 1/21 20060101
C12N001/21 |
Claims
1. A method of delivering one or more siRNAs to animal cells, the
method comprising infecting the animal cells with live invasive
bacteria containing one or more siRNAs or one or more DNA molecules
encoding one or more siRNAs.
2. A method of regulating gene expression in animal cells, the
method comprising infecting the animal cells with live invasive
bacteria containing one or more siRNAs or one or more DNA molecules
encoding one or more siRNAs, wherein the expressed siRNAs interfere
with the mRNA of the gene to be regulated, thereby regulating
expression of said gene.
3. A method of treating or preventing cancer or a cell
proliferation disorder in a mammal, the method comprising
regulating the expression of a gene in a cell known to increase
cell proliferation by infecting the cells of the mammal with live
invasive bacteria containing one or more siRNAs or one or more DNA
molecules encoding one or more siRNAs.
4. A live invasive bacterium comprising one or more siRNAs or one
or more DNA molecules encoding one or more siRNAs.
5. A prokaryotic vector comprising a DNA molecule encoding an siRNA
and an RNA-polymerase III compatible promoter or a prokaryotic
promoter.
6. The method of claims 1-3, wherein said live invasive bacterium
is a non-pathogenic or non-virulent bacterium
7. The method of claim 6, wherein said live invasive bacterium is a
therapeutic bacterium.
8. The method of claim 6, wherein said live invasive bacterium is
an attenuated strain selected from a member of the group consisting
of Listeria, Shigella, Salmonella, E. coli, and
Bifidobacteriae.
9. The method of claim 8, wherein said Salmonella strain is an
attenuated strain of the Salmonella typhimurium species.
10. The method of claim 9, wherein said attenuated strain of the
Salmonella typhimurium species is SL 7207 or VNP20009.
11. The method of claim 8, wherein said attenuated E. coli strain
is BM 2710.
12. The method of claims 1-3, wherein said live invasive bacterium
is a member of the group consisting of Yersinia spp., Escherichia
spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas
spp., Franciesella spp., Corynebacterium spp., Citrobacter spp.,
Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp.,
Legionella spp., Rhodococcus spp., Pseudomonas Spp., Helicobacter
spp., Salmonella spp., Vibrio spp., Bacillus spp., Leishmania spp.
and Erysipelothrix spp. which have been genetically engineered to
mimic the invasion properties of Shigella spp., Listeria spp.,
Rickettsia spp., or enteroinvasive E. coli spp
13. The method of claim 1, wherein said animal cell is in vivo or
in vitro.
14. The method of claims 1-2, wherein said animal cell is a
mammalian cell.
15. The method of claim 14, wherein said mammalian cell is a member
of the group consisting of human, bovine, ovine, porcine, feline,
buffalo, canine, goat, equine, donkey, deer, and primate cells.
16. The method of claim 14, wherein said mammalian cell is a human
cell.
17. The method of claim 3, wherein said mammal is a human.
18. The method of claims 1-3, wherein said one or more DNA
molecules encoding said one or more siRNAs are transcribed within
the animal cell.
19. The method of claim 18, wherein said one or more siRNAs are
transcribed within the animal cell as shRNAs.
20. The method of claim 18, wherein said one or more DNA molecules
encoding said one or more siRNAs comprise an RNA-polymerase III
promoter.
21. The method of claim 20, wherein said RNA-polymerase III
promoter is a U6 promoter or an H1 promoter
22. The method of claims 1-3, wherein said one or more DNA
molecules encoding said one or more siRNAs are transcribed within
the bacterium.
23. The method of claim 22, wherein said one or more DNA molecules
encoding one or more siRNAs comprise a prokaryotic promoter.
24. The method of claim 23, wherein said prokaryotic promoter is a
T7 promoter.
25. The method of claims 1-3, wherein said one or more DNA
molecules are introduced to the cell through type III export or
bacterial lysis.
26. The method of claim 25, wherein said bacterial lysis is
triggered by the addition of an intracellular active
antibiotic.
27. The method of claim 26, wherein said antibiotic is
tetracycline.
28. The method of claim 25, wherein said bacterial lysis is
triggered through bacterial metabolic attenuation.
29. The method of claim 28, wherein said metabolic attenuation is
auxotrophy.
30. The method of claims 1-3, wherein said animal cells are
infected with about 10.sup.3 to 10.sup.11 viable invasive
bacteria.
31. The method of claim 30, wherein said animal cells are infected
with about 10.sup.5 to 10.sup.9 viable invasive bacteria.
32. The method of claims 1-3, wherein said animal cells are
infected at a multiplicity of infection ranging from about 0.1 to
10.sup.6.
33. The method of claim 32, wherein said animal cells are infected
at a multiplicity of infection ranging from about 10.sup.2 to
10.sup.4.
34. The method of claim 3, wherein expressed siRNAs interfere with
the mRNA of the gene to be regulated.
35. The method of claim 2 or 34, wherein the expressed siRNAs
direct the multienzyme complex RNA-induced silencing complex of the
cell to interact with the mRNA of the gene to be regulated.
36. The method of claim 35, wherein said complex degrades said
mRNA
37. The method of claim 35, wherein expression of the gene is
decreased or inhibited.
38. The method of claims 2-3, wherein said gene is ras or
.beta.-catenin.
39. The method of claim 38, wherein said ras is k-Ras
40. The method of claim 3, wherein said cell is a colon cancer cell
or a pancreatic cancer cell.
41. The method of claim 40, wherein the colon cancer cell is an SW
480 cell.
42. The method of claim 40, wherein the pancreatic cancer cell is a
CAPAN-1 cell.
43. The live invasive bacterium of claim 4, wherein said live
invasive bacterium is a non-pathogenic or non-virulent
bacterium.
44. The live invasive bacterium of claim 43, wherein said live
invasive bacterium is a therapeutic bacterium.
45. A composition comprising the bacterium of claim 4 and a
pharmaceutically acceptable carrier.
46. A eukaryotic host cell comprising the bacterium of claim 4, and
a pharmaceutically acceptable carrier.
47. The bacterium of claim 4, wherein said live invasive bacterium
is an attenuated strain selected from a member of the group
consisting of Listeria, Shigella, Salmonella, E. coli, and
Bifidobacteriae.
48. The bacterium of claim 47, wherein said Salmonella strain is an
attenuated strain of the Salmonella typhimurium species.
49. The bacterium of claim 48, wherein said attenuated strain of
the Salmonella typhimurium species is SL 7207 or VNP20009.
50. The bacterium of claim 47, wherein said attenuated E. coli
strain is BM 2710.
51. The prokaryotic vector of claim 5, wherein the RNA-polymerase
III promoter is a U6 promoter or an H1 promoter.
52. The prokaryotic vector of claim 5, wherein the prokaryotic
promoter is a T7 promoter
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation application of U.S. Ser.
No. 11/793,429, filed Nov. 20, 2007, which is a 35 U.S.C. .sctn.371
National Phase Application of PCT/US2005/045513, filed Dec. 16,
2005; which claims the benefit of, and priority to, U.S. Ser. No.
60/637,277 filed Dec. 17, 2004 and U.S. Ser. No. 60/651,238 filed
Feb. 8, 2005, each of which is incorporated by reference in its
entirety.
BACKGROUND
[0002] Gene silencing through RNAi (RNA-interference) by use of
short interfering RNA (siRNA) has emerged as a powerful tool for
molecular biology and holds the potential to be used for
therapeutic gene silencing. Short hairpin RNA (shRNA) transcribed
from small DNA plasmids within the target cell has also been shown
to mediate stable gene silencing and achieve gene knockdown at
levels comparable to those obtained by transfection with chemically
synthesized siRNA (T. R. Brummelkamp, R. Bernards, R. Agami,
Science 296, 550 (2002), P. J. Paddison, A. A. Caudiy, G. J.
Hannon, PNAS 99, 1443 (2002)).
[0003] Possible applications of RNAi for therapeutic purposes are
extensive and include silencing and knockdown of disease genes such
as oncogenes or viral genes. One major obstacle for the therapeutic
use of RNAi is the delivery of siRNA to the target cell (Zamore P
D, Aronin N. Nature Medicine 9, (3):266-8 (2003)). In fact,
delivery has been described as the major hurdle now for RNAi
(Phillip Sharp, cited by Nature news feature, Vol 425, 2003,
10-12)
[0004] Two methods have been described which can be used in mouse
models:
[0005] (1) Direct hydrodynamic intravenous injection of siRNA or
shRNA-encoding plasmids: using this method, several authors have
described application of RNAi against various conditions, e.g.
hepatitis B (A. P. McCaffrey et al., Nat. Biotechnol. 2003 June;
21(6):639-44), fulminant hepatitis (E. Song, S. K. Lee, J. Wang, N.
Ince, J. MM, J. Chen, P. Shankar, J. Lieberman. Nature Medicine 9,
347 (2003)), tumor xenograft (Spaenkuch B, et al. JNCI, 96(1):
862-72 (2004)), hepatic transgene expression (D. L. Lewis, J. E.
Hagstrom, A. G. Loomis, J. A. Wolff, H. Hereijer, Nature Genetics,
32, 107 (2002), D. R. Sorensen D R, M. Leirdal, M. Sioud, JMB, 327,
761 (2003)). This method uses a high pressure and high volume
injection (2.5 ml) into the mouse tail vein. The mechanism of
siRNA/DNA uptake into the cells is not clear but probably
mechanical damage to the vascular endothelial layer is involved. A
clear disadvantage of this method is that this is not a method
which could be developed into human application as it involves a
massive volume charge and completely unknown mechanism of
action.
[0006] (2) Direct injection into the target tissue (brain) of an
siRNA encoding adenoviral vector (H. Xia, Q. Mao, H. L. Paulson, B.
L. Davidson, Nat Biotechnol, 20, 1006 (2002)). This method showed
silencing of transgene (GFP) expression in the brain tissues
reached by the adenoviral vector. However, the area of silencing
could not be predicted reliably. This method might be developed
further and might become applicable for local, e.g. intratumoral
injection. Viral vectors have been used widely for gene therapy
purposes, but one lesson learned from gene therapy experiments is
that viral spreading can be unpredictable at times and lead to
unwanted side effects (Marshall E. Science 286(5448): 2244-5
(1999)). A new method is needed for the safe and predictable
administration of interfering RNAs to mammals.
SUMMARY OF THE INVENTION
[0007] The invention generally pertains to methods of delivering
one or more siRNAs to a eukaryotic cell by introducing a bacterium
to the cell, wherein the bacterium contains one or more siRNAs or
one or more DNA molecules encoding one or more siRNAs.
[0008] In one embodiment of this method, the eukaryotic cell is in
vivo. In another embodiment of this invention, the eukaryotic cell
is in vitro.
[0009] The invention also pertains to a method of regulating gene
expression in a eukaryotic cell, by introducing a bacterium to the
cell, wherein the bacterium contains one or more siRNAs or one or
more DNA molecules encoding one or more siRNAs, wherein the
expressed siRNAs interfere with the mRNA of the gene to be
regulated, thereby regulating expression of the gene.
[0010] In one embodiment of this method, the expressed siRNAs
direct the multienzyme complex RISC(RNA-induced silencing complex)
of the cell to interact with the mRNA to be regulated. This complex
degrades the mRNA. This causes the expression of the gene to be
decreased or inhibited. In another embodiment of this method, the
gene is ras or .beta.-catenin. In one aspect of this embodiment,
the ras is k-Ras.
[0011] In one embodiment of the above methods of the invention, the
eukaryotic cell is a mammalian cell. In one aspect of this
embodiment, the mammalian cell is a human cell.
[0012] The invention also pertains to a method of treating or
preventing cancer or a cell proliferation disorder in a mammal, by
regulating the expression of a gene or several genes in a cell
known to increase cell proliferation by introducing a bacterium to
the cell. The bacterium contains one or more siRNAs or one or more
DNA molecules encoding one or more siRNAs.
[0013] In one embodiment of this method of the invention, the
mammal is a human. In another embodiment of this method the
expressed siRNAs interfere with the mRNA of the gene to be
regulated. In one aspect of this embodiment, the expressed siRNAs
direct the multienzyme complex RISC(RNA-induced silencing complex)
of the cell to interact with the mRNA to be regulated. This complex
degrades the mRNA. This causes the expression of the gene to be
decreased or inhibited.
[0014] In another embodiment of this method, the gene is ras or
.beta.-catenin. In one aspect of this embodiment, the ras is
k-Ras.
[0015] In another embodiment of this method of the invention, the
cell is a colon cancer cell or a pancreatic cancer cell. In one
aspect of this embodiment, the colon cancer cell is an SW 480 cell.
In another aspect of this embodiment, the pancreatic cancer cell is
a CAPAN-1 cell.
[0016] In one embodiment of the above methods of the invention, the
bacterium is non-pathogenic or non-virulent. In another aspect of
this embodiment, the bacterium is therapeutic. In another aspect of
this embodiment, the bacterium is an attenuated strain selected
from the group consisting of Listeria, Shigella, Salmonella, E.
coli, and Bifidobacteriae. Optionally, the Salmonella strain is an
attenuated strain of the Salmonella typhimurium species.
Optionally, the Salmonella typhimurium strain is SL 7207 or
VNP20009. Optionally, the E. coli strain is BM 2710.
[0017] In another embodiment of the above methods of the invention,
the one or more DNA molecules encoding the one or more siRNAs are
transcribed within the eukaryotic cell. In one aspect of this
embodiment, the one or more siRNAs are transcribed within the
eukaryotic cells as shRNAs. In another aspect of this embodiment,
the one or more DNA molecules encoding the one or more siRNAs
contains an RNA-polymerase III promoter. Optionally, the RNA
polymerase III promoter is a U6 promoter or an H1 promoter.
[0018] In another embodiment of the above methods of the invention,
the one or more DNA molecules encoding one or more siRNAs are
transcribed within the bacterium. In one aspect of this embodiment,
the one or more DNA molecules contain a prokaryotic promoter.
Optionally, the prokaryotic promoter is a T7 promoter.
[0019] In another embodiment of the above methods of the invention,
the one or more DNA molecules are introduced to the eukaryotic cell
through type III export or bacterial lysis. In one aspect of this
embodiment, the bacterial lysis is triggered by the addition of an
intracellular active antibiotic. Optionally, the antibiotic is
tetracycline. In another aspect of this embodiment, the bacterial
lysis is triggered through bacterial metabolic attenuation.
Optionally, the metabolic attenuation is auxotrophy.
[0020] The invention also pertains to a bacterium containing one or
more siRNAs or one or more DNA molecules encoding one or more
siRNAs.
[0021] In one embodiment of this invention, the bacterium is a
non-pathogenic or a non-virulent bacterium. In another aspect of
this embodiment, the bacterium is a therapeutic bacterium.
[0022] In another embodiment of this invention, the bacterium is an
attenuated strain selected from a member of the group consisting of
Listeria, Shigella, Salmonella, E. coli, and Bifidobacteriae.
Optionally, the Salmonella strain is an attenuated strain of the
Salmonella typhimurium species. Optionally, the Salmonella
typhimurium strain is SL 7207 or VNP20009. Optionally, the E. coli
strain is BM 2710.
[0023] The invention also pertains to a prokaryotic vector
containing a DNA encoding one or more siRNAs and an RNA-polymerase
III compatible promoter or a prokaryotic promoter.
[0024] In one embodiment of this vector of the invention, the RNA
polymerase III promoter is a U6 promoter or an H1 promoter. In
another embodiment of this vector of the invention, the prokaryotic
promoter is a T7 promoter.
[0025] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference. In the case of
conflict, the present specification, including definitions, will
control. In addition, the materials, methods, and examples are
illustrative only and are not intended to be limiting.
[0026] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A shows micrographs of invasion of SL 7207 into SW 480
cells. FIG. 1B shows FACS analysis of a knockdown of green
fluorescent protein expression in CRL 2583 cells. FIG. 1C shows
micrographs showing loss of fluorescence in CRL 2583 cells.
[0028] FIG. 2A shows Western blots of k-Ras and .beta.-catenin in
SW 480 cells. FIG. 2B is a series of bar charts showing viability
of SW 480 cells under various treatment regimens. FIG. 2C shows
photographs of tumorigenicity in nude mice injected with SW 480
cells transfected with various siRNAs.
[0029] FIG. 3A shows micrographs of transgenic mouse liver
sections. FIG. 3B shows flow cytometry measurements of hepatocyte
and splenocyte suspensions.
[0030] FIG. 4 shows Western blots of k-Ras and .beta.-catenin from
SW 480 cells transfected with silencer plasmid.
[0031] FIG. 5 shows micrographs of histochemical staining of liver
sections of mice shoing changes in GFP expression levels.
[0032] FIG. 6A is a schematic showing the Transkingdom RNA
Interference Plasmid (TRIP). FIG. 6B is a photograph showing an
immunoblot of .beta.-catenin from SW 480 cells transfected with
TRIP. FIG. 6C is a photograph showing an immunoblot of
.beta.-catenin from SW 480 cells transfected with TRIP for exposure
time from 30 to 120 minutes. FIG. 6D shows an RT-PCR photograph of
.beta.-catenin and k-Ras mRNA from SW 480 cells transfected with
TRIP. FIG. 6E is a photograph showing an immunoblot of k-Ras in SW
480 and DLD1 cells following transfection with a TRIP against
mutant k-Ras (GGT.fwdarw.GTT at codon 12) mutant k-Ras
(GGC.fwdarw.GAC at codon 13. FIG. 6F is a photograph showing an
immunoblot of SW 480 cells transfected with a TRIP against wild
type k-Ras.
[0033] FIG. 7A is a photograph of RT-PCR showing .beta.-catenin
silencing following treatment with E. coli expressed shRNAs. FIG.
7B is schematic showing specific cleavage sites in .beta.-catenin.
FIG. 7C is a photograph of a 5'-RACE-PCR showing specific cleavage
products. FIG. 7D is a photograph of a blot showing the mRNA
expression of various genes.
[0034] FIG. 8A is a photograph of cellular staining showing that
both Inv and Hly are required for bacterial entry. FIG. 8B is a
photograph of an RNA blot showing that TRIP lacking Hly is unable
to induce knockdown of a target gene. FIG. 8C is a photograph of an
RNA blot showing that both Inv and Hly are required to facilitate
efficient transkingdom iRNA. FIG. 8D is a photograph of an RNA blot
showing the effect of delayed addition of tetracycline on gene
silencing. FIG. 8E is a photograph of cellular staining showing
lack of significant bacterial replication in the absence of
antibiotics beyond 2 h incubation.
[0035] FIG. 9A is a graph showing that oral administration of E.
coli expressing shRNA against .beta.-catenin in mice leads to
significant reduction of .beta.-catenin expression in the
intestinal epithelium. FIG. 9B is a photograph of
immunohistochemistry staining of intestinal epithelium with or
without treatment. FIG. 9C is a graph showing a decrease in
.beta.-catenin mRNA expression following treatment. FIG. 9D is a
graph showing a decrease in .beta.-catenin protein expression
following treatment. FIG. 9E is a photograph of
immunohistochemistry staining showing decrease in .beta.-catenin
protein expression following treatment.
[0036] FIG. 10 is a photograph of immunohistochemistry staining
showing that GAPDH expression is not altered by E. coli expressing
shRNA against .beta.-catenin after oral dosing in mice.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The invention pertains to methods of delivering small
interfering RNAs (siRNAs) to eukaryotic cells using non-pathogenic
or therapeutic strains of bacteria. The bacteria deliver RNA
encoding DNA or RNA, itself, to effect RNA interference (RNAi). The
interfering RNA of the invention regulates gene expression in
eukaryotic cells. It silences or knocks down genes of interest
inside target cells. The interfering RNA directs the cell-owned
multienzyme-complex RISC(RNA-induced silencing complex) to the mRNA
of the gene to be silenced. Interaction of RISC and mRNA results in
degradation of the mRNA. This leads to effective
post-transcriptional silencing of the gene of interest. This method
is referred to as Bacteria Mediated Gene Silencing (BMGS).
[0038] Bacterial delivery is more attractive than viral delivery as
it can be controlled by use of antibiotics and attenuated bacterial
strains which are unable to multiply. Also, bacteria are much more
accessible to genetic manipulation which allows the production of
vector strains specifically tailored to certain applications. In
one embodiment of the invention, the methods of the invention are
used to create bacteria which cause RNAi in a tissue specific
manner.
[0039] The siRNA is either introduced into the target cell directly
or by transfection or can be transcribed within the target cell as
hairpin-structured dsRNA (shRNA) from specific plasmids with
RNA-polymerase III compatible promoters (U6, H1) (P. J. Paddison,
A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002), T. R.
Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002)).
[0040] Liberation of siRNA encoding plasmid from the intracellular
bacteria occurs through active mechanisms. One mechanism involves
the type III export system in S. typhimuriumm, a specialised
multiprotein complex spanning the bacterial cell membrane whose
functions include secretion of virulence factors to the outside of
the cell to allow signaling towards the target cell, but which can
also be used to deliver antigens into target cells. (Russmann H.
Int J Med Microbiol, 293:107-12 (2003)) or through bacterial lysis
and liberation of bacterial contents into the cytoplasm. The lysis
of intracellular bacteria is triggered through addition of an
intracellularly active antibiotic (tetracycline) or occurs
naturally through bacterial metabolic attenuation (auxotrophy).
After liberation of the eukaryotic transcription plasmid, shRNA or
siRNA are produced within the target cell and trigger the highly
specific process of mRNA degradation, which results in silencing of
the targeted gene.
[0041] The non-virulent bacteria of the invention have invasive
properties and may enter a mammalian host cell through various
mechanisms. In contrast to uptake of bacteria by professional
phagocytes, which normally results in the destruction of the
bacterium within a specialized lysosome, invasive bacteria strains
have the ability to invade non-phagocytic host cells. Naturally
occurring examples of such bacteria are intracellular pathogens
such as Listeria, Shigella and Salmonella, but this property can
also be transferred to other bacteria such as E. coli and
Bifidobacteriae, including probiotics through transfer of
invasion-related genes (P. Courvalin, S. Goussard, C.
Grillot-Courvalin, C.R. Acad. Sci. Paris 318,1207 (1995)). In other
embodiments of the invention, bacteria used to deliver interfering
RNAs to host cells include Shigella flexneri (D. R. Sizemore, A. A.
Branstrom, J. C. Sadoff, Science 270, 299 (1995)), invasive E. coli
(P. Courvalin, S. Goussard, C. Grillot-Courvalin, C.R. Acad. Sci.
Paris 318,1207 (1995), C. Grillot-Courvalin, S. Goussard, F. Huetz,
D. M. Ojcius, P. Courvalin, Nat Biotechnol 16, 862 (1998)),
Yersinia enterocolitica (A. Al-Mariri A, A. Tibor, P. Lestrate, P.
Mertens, X. De Bolle, J. J. Letesson Infect Immun 70, 1915 (2002))
and Listeria monocytogenes (M. Hense, E. Domann, S. Krusch, P.
Wachholz, K. E. Dittmar, M. Rohde, J. Wehland, T. Chakraborty, S.
Weiss, Cell Microbiol 3, 599 (2001), S. Pilgrim, J. Stritzker, C.
Schoen, A. Kolb-Maurer, G. Geginat, M. J. Loessner, I. Gentschev,
W. Goebel, Gene Therapy 10, 2036 (2003)). Any invasive bacterium is
useful for DNA transfer into eukaryotic cells (S. Weiss, T.
Chakraborty, Curr Opinion Biotechnol 12, 467 (2001)).
[0042] BMGS is performed using the naturally invasive pathogen
Salmonella typhimurium. In one aspect of this embodiment, the
strains of Salmonella typhimurium include SL 7207 and VNP20009 (S.
K. Hoiseth, B. A. D. Stocker, Nature 291, 238 (1981); Pawelek J M,
Low K B, Bermudes D. Cancer Res. 57(20):4537-44 (Oct. 15 1997)). In
another embodiment of the invention, BMGS is performed using
attenuated E. coli. In one aspect of this embodiment, the strain of
E. coli is BM 2710 (C. Grillot-Courvalin, S. Goussard, F. Huetz, D.
M. Ojcius, P. Courvalin, Nat Biotechnol 16, 862 (1998)). In another
aspect of this embodiment, the BM 2710 strain is engineered to
possess cell-invading properties through an invasion plasmid. In
one aspect of the invention, this plasmid is pGB2inv-hly.
[0043] A double "trojan horse" technique is also used with an
invasive and auxotrophic bacterium carrying a eukaryotic
transcription plasmid. This plasmid is, in turn, transcribed by the
target cell to form a hairpin RNA structure that triggers the
intracellular process of RNAi. This method of the invention induces
significant gene silencing of a variety of genes. In certain
aspects of this embodiment, the genes include a transgene (GFP), a
mutated oncogene (k-Ras) and a cancer related gene (.beta.-catenin)
in vitro.
[0044] The invention also pertains to a variation of the described
method, termed Bacteria Transcribed Gene Silencing (BTGS). In this
aspect of the invention, siRNA is directly produced by the invasive
bacteria as opposed to the target cell. A transcription plasmid
controlled by a prokaryotic promoter (e.g. T7) is inserted into the
carrier bacteria through standard transformation protocols. siRNA
is produced within the bacteria and is liberated within the
mammalian target cell after bacterial lysis triggered either by
auxotrophy or by timed addition of antibiotics.
[0045] The RNAi methods of the invention, including BMGS and BTGS
are used as a cancer therapy or to prevent cancer. This method is
effected by silencing or knocking down genes involved with cell
proliferation or other cancer phenotypes. Examples of these genes
are k-Ras and .beta.-catenin. Specifically, k-Ras and
.beta.-catenin are targets for RNAi based therapy of colon cancer.
These oncogenes are active and relevant in the majority of clinical
cases. BMGS is applied to reach the intestinal tract for colon
cancer treatment and prevention. These methods are also used to
treat of animals carrying xenograft tumors, to treat and prevent
cancer in k-Ras V12 model of intestinal tumorgenesis, and to
prevent and treat tumors in the adenomatous polyposis coli min
mouse model (APC-min model) In this model, the mouse has a
defective APC gene resulting in the formation of numerous
intestinal and colonic polyps which is used as an animal model for
human familiar adenomatous polyposis coli (FAP) of intestinal
tumorigenesis.
[0046] The invention also encompasses a prokaryotic shRNA-encoding
transcription plasmid for use with invasive bacteria to perform
Bacteria-Transcribed Gene Silencing (BTGS). These plasmids are used
to screen different cancer-related targets in transgenic as well as
wild type animals for therapeutic experiments.
[0047] The RNAi methods of the invention, including BMGS and BTGS
are also used to treat or prevent viral diseases (e.g. hepatitis)
and genetic disorders.
[0048] The RNAi methods of the invention, including BMGS and BTGS
are also used to create cancer-preventing "probiotic bacteria" for
use, especially with the target of GI tract or liver.
[0049] The RNAi methods of the invention, including BMGS and BTGS
are used as therapy against inflammatory conditions, e.g.
hepatitis, inflammatory bowel disease (IBD) or colitis. These
methods are used to silence or knockdown non-cancer gene targets
(viral genes, for treatment and prevention of hepatitis B, C;
inflammatory genes, for treatment and prevention of inflammatory
bowel disease) and others.
[0050] The RNAi methods of the invention, including BMGS and BTGS
are used to create transient "knockdown" genetic animal models as
opposed to genetically engineered knockout models to discover gene
functions. The methods are also used as in vitro transfection tool
for research and drug development
[0051] These methods use bacteria with desirable properties
(invasiveness, attenuation, steerability) for example,
Bifidobacteria and Listeria, are used to perform BMGS and BTGS.
Invasiveness as well as eukaryotic or prokaryotic transcription of
one or several shRNA is conferred to a bacterium using
plasmids.
[0052] The RNAi methods of the invention, including BMGS and BTGS
are used for delivery of gene silencing to the gut and colon, and
for oral application in the treatment of various diseases, namely
colon cancer treatment and prevention. In another aspect of this
embodiment, delivery of gene silencing is extra-intestinal.
[0053] 1. Bacteria Delivering RNA to Eukaryotic Cells
[0054] According to the invention, any microorganism which is
capable of delivering a molecule, e.g., an RNA molecule, into the
cytoplasm of a target cell, such as by traversing the membrane and
entering the cytoplasm of a cell, can be used to deliver RNA to
such cells. In a preferred embodiment, the microorganism is a
prokaryote. In an even more preferred embodiment, the prokaryote is
a bacterium. Also within the scope of the invention are
microorganisms other than bacteria which can be used for delivering
RNA to a cell. For example, the microorganism can be a fungus,
e.g., Cryptococcus neoformans, protozoan, e.g., Trypanosoma cruzi,
Toxoplasma gondii, Leishmania donovani, and plasmodia.
[0055] As used herein, the term "invasive" when referring to a
microorganism, e.g., a bacterium, refers to a microorganism which
is capable of delivering at least one molecule, e.g., an RNA or
RNA-encoding DNA molecule, to a target cell. An invasive
microorganism can be a microorganism which is capable of traversing
a cell membrane, thereby entering the cytoplasm of said cell, and
delivering at least some of its content, e.g., RNA or RNA-encoding
DNA, into the target cell. The process of delivery of the at least
one molecule into the target cell preferably does not significantly
modify the invasion apparatus.
[0056] In a preferred embodiment, the microorganism is a bacterium.
A preferred invasive bacterium is a bacterium which is capable of
delivering at least one molecule, e.g., an RNA or RNA-encoding DNA
molecule, to a target cells, such as by entering the cytoplasm of a
eukaryotic cell. Preferred invasive bacteria are live bacteria,
e.g., live invasive bacteria.
[0057] Invasive microorganisms include microorganisms that are
naturally capable of delivering at least one molecule to a target
cell, such as by traversing the cell membrane, e.g., a eukaryotic
cell membrane, and entering the cytoplasm, as well as
microorganisms which are not naturally invasive and which have been
modified, e.g., genetically modified, to be invasive. In another
preferred embodiment, a microorganism which is not naturally
invasive can be modified to become invasive by linking the
bacterium to an "invasion factor", also termed "entry factor" or
"cytoplasm-targeting factor". As used herein, an "invasion factor"
is a factor, e.g., a protein or a group of proteins which, when
expressed by a non-invasive bacterium, render the bacterium
invasive. As used herein, an "invasion factor" is encoded by a
"cytoplasm-targeting gene".
[0058] Naturally invasive microorganisms, e.g., bacteria, may have
a certain tropism, i.e., preferred target cells. Alternatively,
microorganisms, e.g., bacteria can be modified, e.g., genetically,
to mimic the tropism of a second microorganism.
[0059] Delivery of at least one molecule into a target cell can be
determined according to methods known in the art. For example, the
presence of the molecule, by the decrease in expression of an RNA
or protein silenced thereby, can be detected by hybridization or
PCR methods, or by immunological methods which may include the use
of an antibody.
[0060] Determining whether a microorganism is sufficiently invasive
for use in the invention may include determining whether sufficient
RNA, was delivered to host cells, relative to the number of
microorganisms contacted with the host cells. If the amount of RNA,
is low relative to the number of microorganisms used, it may be
desirable to further modify the microorganism to increase its
invasive potential.
[0061] Bacterial entry into cells can be measured by various
methods. Intracellular bacteria survive treatment by aminoglycoside
antibiotics, whereas extracellular bacteria are rapidly killed. A
quantitative estimate of bacterial uptake can be achieved by
treating cell monolayers with the antibiotic gentamicin to
inactivate extracellular bacteria, then by removing said antibiotic
before liberating the surviving intracellular organisms with gentle
detergent and determining viable counts on standard bacteriological
medium. Furthermore, bacterial entry into cells can be directly
observed, e.g., by thin-section-transmission electron microscopy of
cell layers or by immunofluorescent techniques (Falkow et al.
(1992) Annual Rev. Cell Biol. 8:333). Thus, various techniques can
be used to determine whether a specific bacteria is capable of
invading a specific type of cell or to confirm bacterial invasion
following modification of the bacteria, such modification of the
tropism of the bacteria to mimic that of a second bacterium.
[0062] Bacteria that can be used for delivering RNA according to
the method of the invention are preferably non-pathogenic. However,
pathogenic bacteria can also be used, so long as their
pathogenicity has been attenuated, to thereby render the bacteria
non-harmful to a subject to which it is administered. As used
herein, the term "attenuated bacterium" refers to a bacterium that
has been modified to significantly reduce or eliminate its
harmfulness to a subject. A pathogenic bacterium can be attenuated
by various methods, set forth below.
[0063] Without wanting to be limited to a specific mechanism of
action, the bacterium delivering the RNA into the eukaryotic cell
can enter various compartments of the cell, depending on the type
of bacterium. For example, the bacterium can be in a vesicle, e.g.,
a phagocytic vesicle. Once inside the cell, the bacterium can be
destroyed or lysed and its contents delivered to the eukaryotic
cell. A bacterium can also be engineered to express a phagosome
degrading enyzme to allow leakage of RNA from the phagosome. In
some embodiments, the bacterium can stay alive for various times in
the eukaryotic cell and may continue to produce RNA. The RNA or
RNA-encoding DNA can then be released from the bacterium into the
cell by, e.g., leakage. In certain embodiments of the invention,
the bacterium can also replicate in the eukaryotic cell. In a
preferred embodiment, bacterial replication does not kill the host
cell. The invention is not limited to delivery of RNA or
RNA-encoding DNA by a specific mechanism and is intended to
encompass methods and compositions permitting delivery of RNA or
RNA-encoding DNA by a bacterium independently of the mechanism of
delivery.
[0064] Set forth below are examples of bacteria which have been
described in the literature as being naturally invasive (section
1.1), as well as bacteria which have been described in the
literature as being naturally non-invasive bacteria (section 1.2),
as well as bacteria which are naturally non-pathogenic or which are
attenuated. Although some bacteria have been described as being
non-invasive (section 1.2), these may still be sufficiently
invasive for use according to the invention. Whether traditionally
described as naturally invasive or non-invasive, any bacterial
strain can be modified to modulate, in particular to increase, its
invasive characteristics (e.g., as described in section 1.3).
[0065] 1.1 Naturally Invasive Bacteria
[0066] The particular naturally invasive bacteria employed in the
present invention is not critical thereto. Examples of such
naturally-occurring invasive bacteria include, but are not limited
to, Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp.,
and enteroinvasive Escherichia coli.
[0067] The particular Shigella strain employed is not critical to
the present invention. Examples of Shigella strains which can be
employed in the present invention include Shigella flexneri 2a
(ATCC No. 29903), Shigella sonnei (ATCC No. 29930), and Shigella
disenteriae (ATCC No. 13313). An attenuated Shigella strain, such
as Shigella flexneri 2a 2457T aroA virG mutant CVD 1203 (Noriega et
al. supra), Shigella flexneri M90T icsA mutant (Goldberg et al.
Infect. Immun., 62:5664-5668 (1994)), Shigella flexneri Y SFL114
aroD mutant (Karnell et al. Vacc., 10:167-174 (1992)), and Shigella
flexneri aroA aroD mutant (Verma et al. Vacc., 9:6-9 (1991)) are
preferably employed in the present invention. Alternatively, new
attenuated Shigella spp. strains can be constructed by introducing
an attenuating mutation either singularly or in conjunction with
one or more additional attenuating mutations.
[0068] At least one advantage to Shigella RNA vaccine vectors is
their tropism for lymphoid tissue in the colonic mucosal surface.
In addition, the primary site of Shigella replication is believed
to be within dendritic cells and macrophages, which are commonly
found at the basal lateral surface of M cells in mucosal lymphoid
tissues (reviewed by McGhee, J. R. et al. (1994) Reproduction,
Fertility, & Development 6:369; Pascual, D. W. et al. (1994)
Immunomethods 5:56). As such, Shigella vectors may provide a means
to express antigens in these professional antigen presenting cells.
Another advantage of Shigella vectors is that attenuated Shigella
strains deliver nucleic acid reporter genes in vitro and in vivo
(Sizemore, D. R. et al. (1995) Science 270:299; Courvalin, P. et
al. (1995) Comptes Rendus de 1 Academie des Sciences Serie
III-Sciences de la Vie-Life Sciences 318:1207; Powell, R. J. et al.
(1996) In: Molecular approaches to the control of infectious
diseases. F. Brown, E. Norrby, D. Burton and J. Mekalanos, eds.
Cold Spring Harbor Laboratory Press, New York. 183; Anderson, R. J.
et al. (1997) Abstracts for the 97th General Meeting of the
American Society for Microbiology:E.). On the practical side, the
tightly restricted host specificity of Shigella stands to prevent
the spread of Shigella vectors into the food chain via intermediate
hosts. Furthermore, attenuated strains that are highly attenuated
in rodents, primates and volunteers have been developed (Anderson
et al. (1997) supra; Li, A. et al. (1992) Vaccine 10:395; Li, A. et
al. (1993) Vaccine 11:180; Karnell, A. et al. (1995) Vaccine 13:88;
Sansonetti, P. J. and J. Arondel (1989) Vaccine 7:443; Fontaine, A.
et al. (1990) Research in Microbiology 141:907; Sansonetti, P. J.
et al. (1991) Vaccine 9:416; Noriega, F. R. et al. (1994) Infection
& Immunity 62:5168; Noriega, F. R. et al. (1996) Infection
& Immunity 64:3055; Noriega, F. R. et al. (1996) Infection
& Immunity 64:23; Noriega, F. R. et al. (1996) Infection &
Immunity 64:3055; Kotloff, K. L. et al. (1996) Infection &
Immunity 64:4542). This latter knowledge will allow the development
of well tolerated Shigella vectors for use in humans.
[0069] Attenuating mutations can be introduced into bacterial
pathogens using non-specific mutagenesis either chemically, using
agents such as N-methyl-N'-nitro-N-nitrosoguanidine, or using
recombinant DNA techniques; classic genetic techniques, such as
Tn10 mutagenesis, P22-mediated transduction, .lamda., phage
mediated crossover, and conjugational transfer; or site-directed
mutagenesis using recombinant DNA techniques. Recombinant DNA
techniques are preferable since strains constructed by recombinant
DNA techniques are far more defined. Examples of such attenuating
mutations include, but are not limited to:
[0070] (i) auxotrophic mutations, such as aro (Hoiseth et al.
Nature, 291:238-239 (1981)), gua (McFarland et al. Microbiol.
Path., 3:129-141 (1987)), nad (Park et al. J. Bact., 170:3725-3730
(1988), thy (Nnalue et al. Infect. Immun., 55:955-962 (1987)), and
asd (Curtiss, supra) mutations;
[0071] (ii) mutations that inactivate global regulatory functions,
such as cya (Curtiss et al. Infect. Immun., 55:3035-3043 (1987)),
crp (Curtiss et al (1987), supra), phoP/phoQ (Groisman et al. Proc.
Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et al. Proc.
Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phop.sup.c (Miller et
al. J. Bact., 172:2485-2490 (1990)) or ompR (Dorman et al. Infect.
Immun, 57:2136-2140 (1989)) mutations;
[0072] (iii) mutations that modify the stress response, such as
recA (Buchmeier et al. Mol. Micro., 7:933-936 (1993)), htrA
(Johnson et al. Mol. Micro., 5:401-407 (1991)), htpR (Neidhardt et
al. Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp
(Neidhardt et al. Ann. Rev. Genet., 18:295-329 (1984)) and groEL
(Buchmeier et al. Sci., 248:730-732 (1990)) mutations;
[0073] (iv) mutations in specific virulence factors, such as IsyA
(Libby et al. Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag
or prg (Miller et al (1990), supra; and Miller et al (1989),
supra), iscA or virG (d'Hauteville et al. Mol. Micro., 6:833-841
(1992)), plcA (Mengaud et al. Mol. Microbiol., 5:367-72 (1991);
Camilli et al. J. Exp. Med, 173:751-754 (1991)), and act (Brundage
et al. Proc. Natl. Acad. Sci., USA, 90:11890-11894 (1993))
mutations;
[0074] (v) mutations that affect DNA topology, such as topA (Galan
et al. Infect. Immun., 58:1879-1885 (1990));
[0075] (vi) mutations that disrupt or modify the cell cycle, such
as min (de Boer et al. Cell, 56:641-649 (1989)).
[0076] (vii) introduction of a gene encoding a suicide system, such
as sacB (Recorbet et al. App. Environ. Micro., 59:1361-1366 (1993);
Quandt et al. Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al. App.
Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA
(Molin et al. Ann Rev. Microbiol., 47:139-166 (1993));
[0077] (viii) mutations that alter the biogenesis of
lipopolysaccharide and/or lipid A, such as rFb (Raetz in Esherishia
coli and Salmonella typhimurium, Neidhardt et al., Ed., ASM Press,
Washington D.C. pp 1035-1063 (1996)), galE (Hone et al. J. Infect.
Dis., 156:164-167 (1987)) and htrB (Raetz, supra), msbB (Reatz,
supra)
[0078] (ix) introduction of a bacteriophage lysis system, such as
lysogens encoded by P22 (Rennell et al. Virol, 143:280-289 (1985)),
.lamda., murein transglycosylase (Bienkowska-Szewczyk et al. Mol.
Gen. Genet., 184:111-114 (1981)) or S-gene (Reader et al. Virol,
43:623-628 (1971)); and
[0079] The attenuating mutations can be either constitutively
expressed or under the control of inducible promoters, such as the
temperature sensitive heat shock family of promoters (Neidhardt et
al. supra), or the anaerobically induced nirB promoter (Harborne et
al. Mol. Micro., 6:2805-2813 (1992)) or repressible promoters, such
as uapA (Gorfinkiel et al. J. Biol. Chem., 268:23376-23381 (1993))
or gcv (Stauffer et al. J. Bact., 176:6159-6164 (1994)).
[0080] The particular Listeria strain employed is not critical to
the present invention. Examples of Listeria strains which can be
employed in the present invention include Listeria monocytogenes
(ATCC No. 15313). Attenuated Listeria strains, such as L.
monocytogenes actA mutant (Brundage et al. supra) or L.
monocytogenes plcA (Camilli et al. J. Exp. Med., 173:751-754
(1991)) are preferably used in the present invention.
Alternatively, new attenuated Listeria strains can be constructed
by introducing one or more attenuating mutations in groups (i) to
(vii) as described for Shigella spp. above.
[0081] The particular Salmonella strain employed is not critical to
the present invention. Examples of Salmonella strains which can be
employed in the present invention include Salmonella typhi (ATCC
No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated
Salmonella strains are preferably used in the present invention and
include S. typhi-aroC-aroD (Hone et al. Vacc. 9:810 (1991) and S.
typhimurium-aroA mutant (Mastroeni et al. Micro. Pathol. 13:477
(1992)). Alternatively, new attenuated Salmonella strains can be
constructed by introducing one or more attenuating mutations as
described fro Shigella spp. above.
[0082] The particular Rickettsia strain employed is not critical to
the present invention. Examples of Rickettsia strains which can be
employed in the present invention include Rickettsia Rickettsiae
(ATCC Nos. VR149 and VR891), Ricketsia prowaseckii (ATCC No.
VR233), Rickettsia tsutsugamuchi (ATCC Nos. VR312, VR150 and
VR609), Rickettsia mooseri (ATCC No. VR144), Rickettsia sibirica
(ATCC No. VR151), and Rochalimaea quitana (ATCC No. VR358).
Attenuated Rickettsia strains are preferably used in the present
invention and can be constructed by introducing one or more
attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0083] The particular enteroinvasive Escherichia strain employed is
not critical to the present invention. Examples of enteroinvasive
Escherichia strains which can be employed in the present invention
include Escherichia coli strains 4608-58, 1184-68, 53638-C-17,
13-80, and 6-81 (Sansonetti et al. Ann. Microbiol. (Inst. Pasteur),
132A:351-355 (1982)). Attenuated enteroinvasive Escherichia strains
are preferably used in the present invention and can be constructed
by introducing one or more attenuating mutations in groups (i) to
(vii) as described for Shigella spp. above.
[0084] Furthermore, since certain microorganisms other than
bacteria can also interact with integrin molecules (which are
receptors for certain invasion factors) for cellular uptake, such
microorganisms can also be used for introducing RNA into target
cells. For example, viruses, e.g., foot-and-mouth disease virus,
echovirus, and adenovirus, and eukaryotic pathogens, e.g.,
Histoplasma capsulatum and Leishmania major interact with integrin
molecules.
[0085] 1.2 Less Invasive Bacteria
[0086] Examples of bacteria which can be used in the invention and
which have been described in the literature as being non-invasive
or at least less invasive than the bacteria listed in the previous
section (1.1) include, but are not limited to, Yersinia spp.,
Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp.,
Aeromonas spp., Franciesella spp., Corynebacterium spp.,
Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp.,
Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas
spp., Helicobacter spp., Vibrio spp., Bacillus spp., and
Erysipelothrix spp. It may be necessary to modify these bacteria to
increase their invasive potential.
[0087] The particular Yersinia strain employed is not critical to
the present invention. Examples of Yersinia strains which can be
employed in the present invention include Y. enterocolitica (ATCC
No. 9610) or Y. pestis (ATCC No. 19428). Attenuated Yersinia
strains, such as Y. enterocolitica Ye03-R2 (al-Hendy et al. Infect.
Immun, 60:870-875 (1992)) or Y. enterocolitica aroA (O'Gaora et al.
Micro. Path., 9:105-116 (1990)) are preferably used in the present
invention. Alternatively, new attenuated Yersinia strains can be
constructed by introducing one or more attenuating mutations in
groups (i) to (vii) as described for Shigella spp. above.
[0088] The particular Escherichia strain employed is not critical
to the present invention. Examples of Escherichia strains which can
be employed in the present invention include E. coli H10407
(Elinghorst et al. Infect. Immun., 60:2409-2417 (1992)), and E.
coli EFC4, CFT325 and CPZ005 (Donnenberg et al. J. Infect. Dis.,
169:831-838 (1994)). Attenuated Escherichia strains, such as the
attenuated turkey pathogen E. coli 02 carAB mutant (Kwaga et al.
Infect. Immun., 62:3766-3772 (1994)) are preferably used in the
present invention. Alternatively, new attenuated Escherichia
strains can be constructed by introducing one or more attenuating
mutations in groups (i) to (vii) as described for Shigella spp.
above.
[0089] The particular Klebsiella strain employed is not critical to
the present invention. Examples of Klebsiella strains which can be
employed in the present invention include K. pneumoniae (ATCC No.
13884). Attenuated Klebsiella strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0090] The particular Bordetella strain employed is not critical to
the present invention. Examples of Bordetella strains which can be
employed in the present invention include B. bronchiseptica (ATCC
No. 19395). Attenuated Bordetella strains are preferably used in
the present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0091] The particular Neisseria strain employed is not critical to
the present invention. Examples of Neisseria strains which can be
employed in the present invention include N. meningitidis (ATCC No.
13077) and N. gonorrhoeae (ATCC No. 19424). Attenuated Neisseria
strains, such as N. gonorrhoeae MS11 aro mutant (Chamberlain et al.
Micro. Path., 15:51-63 (1993)) are preferably used in the present
invention. Alternatively, new attenuated Neisseria strains can be
constructed by introducing one or more attenuating mutations in
groups (i) to (vii) as described for Shigella spp. above.
[0092] The particular Aeromonas strain employed is not critical to
the present invention. Examples of Aeromonas strains which can be
employed in the present invention include A. eucrenophila (ATCC No.
23309). Alternatively, new attenuated Aeromonas strains can be
constructed by introducing one or more attenuating mutations in
groups (i) to (vii) as described for Shigella spp. above.
[0093] The particular Franciesella strain employed is not critical
to the present invention. Examples of Franciesella strains which
can be employed in the present invention include F. tularensis
(ATCC No. 15482). Attenuated Franciesella strains are preferably
used in the present invention, and can be constructed by
introducing one or more attenuating mutations in groups (i) to
(vii) as described for Shigella spp. above.
[0094] The particular Corynebacterium strain employed is not
critical to the present invention. Examples of Corynebacterium
strains which can be employed in the present invention include C.
pseudotuberculosis (ATCC No. 19410). Attenuated Corynebacterium
strains are preferably used in the present invention, and can be
constructed by introducing one or more attenuating mutations in
groups (i) to (vii) as described for Shigella spp. above.
[0095] The particular Citrobacter strain employed is not critical
to the present invention. Examples of Citrobacter strains which can
be employed in the present invention include C. freundii (ATCC No.
8090). Attenuated Citrobacter strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0096] The particular Chlamydia strain employed is not critical to
the present invention. Examples of Chlamydia strains which can be
employed in the present invention include C. pneumoniae (ATCC No.
VR1310). Attenuated Chlamydia strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0097] The particular Hemophilus strain employed is not critical to
the present invention. Examples of Hemophilus strains which can be
employed in the present invention include H. sornnus (ATCC No.
43625). Attenuated Hemophilus strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0098] The particular Brucella strain employed is not critical to
the present invention. Examples of Brucella strains which can be
employed in the present invention include B. abortus (ATCC No.
23448). Attenuated Brucella strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0099] The particular Mycobacterium strain employed is not critical
to the present invention. Examples of Mycobacterium strains which
can be employed in the present invention include M. intracellulare
(ATCC No. 13950) and M. tuberculosis (ATCC No. 27294). Attenuated
Mycobacterium strains are preferably used in the present invention,
and can be constructed by introducing one or more attenuating
mutations in groups (i) to (vii) as described for Shigella spp.
above.
[0100] The particular Legionella strain employed is not critical to
the present invention. Examples of Legionella strains which can be
employed in the present invention include L. pneumophila (ATCC No.
33156). Attenuated Legionella strains, such as a L. pneumophila mip
mutant (Ott, FEMS Micro. Rev., 14:161-176 (1994)) are preferably
used in the present invention. Alternatively, new attenuated
Legionella strains can be constructed by introducing one or more
attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0101] The particular Rhodococcus strain employed is not critical
to the present invention. Examples of Rhodococcus strains which can
be employed in the present invention include R. equi (ATCC No.
6939). Attenuated Rhodococcus strains are preferably used in the
present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0102] The particular Pseudomonas strain employed is not critical
to the present invention. Examples of Pseudomonas strains which can
be employed in the present invention include P. aeruginosa (ATCC
No. 23267). Attenuated Pseudomonas strains are preferably used in
the present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0103] The particular Helicobacter strain employed is not critical
to the present invention. Examples of Helicobacter strains which
can be employed in the present invention include H. mustelae (ATCC
No. 43772). Attenuated Helicobacter strains are preferably used in
the present invention, and can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0104] The particular Salmonella strain employed is not critical to
the present invention. Examples of Salmonella strains which can be
employed in the present invention include Salmonella typhi (ATCC
No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated
Salmonella strains are preferably used in the present invention and
include S. typhi aroC aroD (Hone et al. Vacc., 9:810-816 (1991))
and S. typhimurium aroA mutant (Mastroeni et al. Micro. Pathol,
13:477-491 (1992))). Alternatively, new attenuated Salmonella
strains can be constructed by introducing one or more attenuating
mutations in groups (i) to (vii) as described for Shigella spp.
above.
[0105] The particular Vibrio strain employed is not critical to the
present invention. Examples of Vibrio strains which can be employed
in the present invention include Vibrio cholerae (ATCC No. 14035)
and Vibrio cincinnatiensis (ATCC No. 35912). Attenuated Vibrio
strains are preferably used in the present invention and include V.
cholerae RSI virulence mutant (Taylor et al. J. Infect. Dis.,
170:1518-1523 (1994)) and V. cholerae ctxA, ace, zot, cep mutant
(Waldor et al. J. Infect. Dis., 170:278-283 (1994)). Alternatively,
new attenuated Vibrio strains can be constructed by introducing one
or more attenuating mutations in groups (i) to (vii) as described
for Shigella spp. above.
[0106] The particular Bacillus strain employed is not critical to
the present invention. Examples of Bacillus strains which can be
employed in the present invention include Bacillus subtilis (ATCC
No. 6051). Attenuated Bacillus strains are preferably used in the
present invention and include B. anthracis mutant pX01 (Welkos et
al. Micro. Pathol, 14:381-388 (1993)) and attenuated BCG strains
(Stover et al. Nat., 351:456-460 (1991)). Alternatively, new
attenuated Bacillus strains can be constructed by introducing one
or more attenuating mutations in groups (i) to (vii) as described
for Shigella spp. above.
[0107] The particular Erysipelothrix strain employed is not
critical to the present invention. Examples of Erysipelothrix
strains which can be employed in the present invention include
Erysipelothrix rhusiopathiae (ATCC No. 19414) and Erysipelothrix
tonsillarum (ATCC No. 43339). Attenuated Erysipelothrix strains are
preferably used in the present invention and include E.
rhusiopathiae Kg-1a and Kg-2 (Watarai et al. J. Vet. Med. Sci.,
55:595-600 (1993)) and E. rhusiopathiae ORVAC mutant
(Markowska-Daniel et al. Int. J. Med. Microb. Virol. Parisit.
Infect. Dis., 277:547-553 (1992)). Alternatively, new attenuated
Erysipelothrix strains can be constructed by introducing one or
more attenuating mutations in groups (i) to (vii) as described for
Shigella spp. above.
[0108] 1.3. Methods for Increasing the Invasive Properties of a
Bacterial Strain
[0109] Whether organisms have been traditionally described as
invasive or non-invasive, these organisms can be engineered to
increase their invasive properties, e.g., by mimicking the invasive
properties of Shigella spp., Listeria spp., Rickettsia spp., or
enteroinvasive E. coli spp. For example, one or more genes that
enable the microorganism to access the cytoplasm of a cell, e.g., a
cell in the natural host of said non-invasive bacteria, can be
introduced into the microorganism.
[0110] Examples of such genes referred to herein as
"cytoplasm-targeting genes" include genes encoding the proteins
that enable invasion by Shigella or the analogous invasion genes of
entero-invasive Escherichia, or listeriolysin O of Listeria, as
such techniques are known to result in rendering a wide array of
invasive bacteria capable of invading and entering the cytoplasm of
animal cells (Formal et al. Infect. Immun., 46:465 (1984); Bielecke
et al. Nature, 345:175-176 (1990); Small et al. In:
Microbiology-1986, pages 121-124, Levine et al. Eds., American
Society for Microbiology, Washington, D.C. (1986); Zychlinsky et
al. Molec. Micro., 11:619-627 (1994); Gentschev et al. (1995)
Infection & Immunity 63:4202; Isberg, R. R. and S. Falkow
(1985) Nature 317:262; and Isberg, R. R. et al. (1987) Cell
50:769). Methods for transferring the above cytoplasm-targeting
genes into a bacterial strain are well known in the art. Another
preferred gene which can be introduced into bacteria to increase
their invasive character encodes the invasin protein from Yersinia
pseudotuberculosis, (Leong et al. EMBO J., 9:1979 (1990)). Invasin
can also be introduced in combination with listeriolysin, thereby
further increasing the invasive character of the bacteria relative
to the introduction of either of these genes. The above genes have
been described for illustrative purposes; however, it will be
obvious to those skilled in the art that any gene or combination of
genes, from one or more sources, that participates in the delivery
of a molecule, in particular an RNA or RNA-encoding DNA molecule,
from a microorganism into the cytoplasm of a cell, e.g., an animal
cell, will suffice. Thus, such genes are not limited to bacterial
genes, and include viral genes, such as influenza virus
hemagglutinin HA-2 which promotes endosmolysis (Plank et al. J.
Biol. Chem., 269:12918-12924 (1994)).
[0111] The above cytoplasm-targeting genes can be obtained by,
e.g., PCR amplification from DNA isolated from an invasive
bacterium carrying the desired cytoplasm-targeting gene. Primers
for PCR can be designed from the nucleotide sequences available in
the art, e.g., in the above-listed references and/or in GenBank,
which is publicly available on the internet
(www.ncbi.nlm.nih.gov/). The PCR primers can be designed to amplify
a cytoplasm-targeting gene, a cytoplasm-targeting operon, a cluster
of cytoplasm-targeting genes, or a regulon of cytoplasm-targeting
genes. The PCR strategy employed will depend on the genetic
organization of the cytoplasm-targeting gene or genes in the target
invasive bacteria. The PCR primers are designed to contain a
sequence that is homologous to DNA sequences at the beginning and
end of the target DNA sequence. The cytoplasm-targeting genes can
then be introduced into the target bacterial strain, e.g., by using
Hfr transfer or plasmid mobilization (Miller, A Short Course in
Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1992); Bothwell et al. supra; and Ausubel et
al. supra), bacteriophage-mediated transduction (de Boer, supra;
Miller, supra; and Ausubel et al. supra), chemical transformation
(Bothwell et al. supra; Ausubel et al. supra), electroporation
(Bothwel et al. supra; Ausubel et al. supra; and Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.) and physical
transformation techniques (Johnston et al. supra; and Bothwell,
supra). The cytoplasm-targeting genes can be incorporated into
lysogenic bacteriophage (de Boer et al. Cell, 56:641-649 (1989)),
plasmids vectors (Curtiss et al. supra) or spliced into the
chromosome (Hone et al. supra) of the target strain.
[0112] In addition to genetically engineering bacteria to increase
their invasive properties, as set forth above, bacteria can also be
modified by linking an invasion factor to the bacteria.
Accordingly, in one embodiment, a bacterium is rendered more
invasive by coating the bacterium, either covalently or
non-covalently, with an invasion factor, e.g., the protein invasin,
invasin derivatives, or a fragment thereof sufficient for
invasiveness. In fact, it has been shown that non-invasive
bacterial cells coated with purified invasin from Yersinia
pseudotuberculosis or the carboxyl-terminal 192 amino acids of
invasin are able to enter mammalian cells (Leong et al. (1990) EMBO
J. 9:1979). Furthermore, latex beads coated with the carboxyl
terminal region of invasin are efficiently internalized by
mammalian cells, as are strains of Staphylococcus aureus coated
with antibody-immobilized invasin (reviewed in Isberg and Tran van
Nhieu (1994) Ann. Rev. Genet. 27:395). Alternatively, a bacterium
can also be coated with an antibody, variant thereof, or fragment
thereof which binds specifically to a surface molecule recognized
by a bacterial entry factor. For example, it has been shown that
bacteria are internalized if they are coated with a monoclonal
antibody directed against an integrin molecule, e.g.,
.alpha.5.beta.1, known to be the surface molecule with which the
bacterial invasin protein interacts (Isberg and Tran van Nhieu,
supra). Such antibodies can be prepared according to methods known
in the art. The antibodies can be tested for efficacy in mediating
bacterial invasiveness by, e.g., coating bacteria with the
antibody, contacting the bacteria with eukaryotic cells having a
surface receptor recognized by the antibody, and monitoring the
presence of intracellular bacteria, according to the methods
described above. Methods for linking an invasion factor to the
surface of a bacterium are known in the art and include
cross-linking.
[0113] 2. Target Cells
[0114] The invention provides a method for delivering RNA to any
type of target cell. As used herein, the term "target cell" refers
to a cell which can be invaded by a bacterium, i.e., a cell which
has the necessary surface receptor for recognition by the
bacterium.
[0115] Preferred target cells are eukaryotic cells. Even more
preferred target cells are animal cells. "Animal cells" are defined
as nucleated, non-chloroplast containing cells derived from or
present in multicellular organisms whose taxanomic position lies
within the kingdom animalia. The cells may be present in the intact
animal, a primary cell culture, explant culture or a transformed
cell line. The particular tissue source of the cells is not
critical to the present invention.
[0116] The recipient animal cells employed in the present invention
are not critical thereto and include cells present in or derived
from all organisms within the kingdom animalia, such as those of
the families mammalia, pisces, avian, reptilia.
[0117] Preferred animal cells are mammalian cells, such as humans,
bovine, ovine, porcine, feline, canine, goat, equine, and primate
cells. The most preferred animal cells are human cells.
[0118] In a preferred embodiment, the target cell is in a mucosal
surface. Certain enteric pathogens, e.g., E. coli, Shigella,
Listeria, and Salmonella, are naturally adapted for this
application, as these organisms possess the ability to attach to
and invade host mucosal surfaces (Kreig et al. supra). Therefore,
in the present invention, such bacteria can deliver RNA molecules
or RNA-encoding DNA to cells in the host mucosal compartment.
[0119] Although certain types of bacteria may have a certain
tropism, i.e., preferred target cells, delivery of RNA or
RNA-encoding DNA to a certain type of cell can be achieved by
choosing a bacterium which has a tropism for the desired cell type
or which is modified such as to be able to invade the desired cell
type. Thus, e.g., a bacterium could be genetically engineered to
mimic mucosal tissue tropism and invasive properties, as discussed
above, to thereby allow said bacteria to invade mucosal tissue, and
deliver RNA or RNA-encoding DNA to cells in those sites.
[0120] Bacteria can also be targeted to other types of cells. For
example, bacteria can be targeted to erythrocytes of humans and
primates by modifying bacteria to express on their surface either,
or both of, the Plasmodium vivax reticulocyte binding proteins-1
and -2, which bind specifically to erythrocytes in humans and
primates (Galinski et al. Cell, 69:1213-1226 (1992)). In another
embodiment, bacteria are modified to have on their surface
asialoorosomucoid, which is a ligand for the asilogycoprotein
receptor on hepatocytes (Wu et al. J. Biol. Chem., 263:14621-14624
(1988)). In yet another embodiment, bacteria are coated with
insulin-poly-L-lysine, which has been shown to target plasmid
uptake to cells with an insulin receptor (Rosenkranz et al. Expt.
Cell Res., 199:323-329 (1992)). Also within the scope of the
invention are bacteria modified to have on their surface p60 of
Listeria monocytogenes, which allows for tropism for hepatocytes
(Hess et al. Infect. Immun., 63:2047-2053 (1995)), or a 60 kD
surface protein from Trypanosoma cruzi which causes specific
binding to the mammalian extra-cellular matrix by binding to
heparin, heparin sulfate and collagen (Ortega-Barria et al. Cell,
67:411-421 (1991)).
[0121] Yet in another embodiment, a cell can be modified to become
a target cell of a bacterium for delivery of RNA. Accordingly, a
cell can be modified to express a surface antigen which is
recognized by a bacterium for its entry into the cell, i.e., a
receptor of an invasion factor. The cell can be modified either by
introducing into the cell a nucleic acid encoding a receptor of an
invasion factor, such that the surface antigen is expressed in the
desired conditions. Alternatively, the cell can be coated with a
receptor of an invasion factor. Receptors of invasion factors
include proteins belonging to the integrin receptor superfamily. A
list of the type of integrin receptors recognized by various
bacteria and other microorganisms can be found, e.g., in Isberg and
Tran Van Nhieu (1994) Ann Rev. Genet. 27:395. Nucleotide sequences
for the integrin subunits can be found, e.g., in GenBank, publicly
available on the internet.
[0122] As set forth above, yet other target cells include fish,
avian, and reptilian cells. Examples of bacteria which are
naturally invasive for fish, avian, and reptilian cells are set
forth below.
[0123] Examples of bacteria which can naturally access the
cytoplasm of fish cells include, but are not limited to Aeromonas
salminocida (ATCC No. 33658) and Aeromonas schuberii (ATCC No.
43700). Attenuated bacteria are preferably used in the invention,
and include A. salmonicidia vapA (Gustafson et al. J. Mol. Biol.,
237:452-463 (1994)) or A. salmonicidia aromatic-dependent mutant
(Vaughan et al. Infect. Immun., 61:2172-2181 (1993)).
[0124] Examples of bacteria which can naturally access the
cytoplasm of avian cells include, but are not restricted to,
Salmonella galinarum (ATCC No. 9184), Salmonella enteriditis (ATCC
No. 4931) and Salmonella typhimurium (ATCC No. 6994). Attenuated
bacteria are preferred to the invention and include attenuated
Salmonella strains such as S. galinarum cya crp mutant (Curtiss et
al. (1987) supra) or S. enteritidis aroA aromatic-dependent mutant
CVL30 (Cooper et al. Infect. Immun, 62:4739-4746 (1994)).
[0125] Examples of bacteria which can naturally access the
cytoplasm of reptilian cells include, but are not restricted to,
Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are
preferable to the invention and include, attenuated strains such as
S. typhimuirum aromatic-dependent mutant (Hormaeche et al.
supra).
[0126] The invention also provides for delivery of RNA to other
eukaryotic cells, e.g., plant cells, so long as there are
microorganisms which are capable of invading such cells, either
naturally or after having been modified to become invasive.
Examples of microorganisms which can invade plant cells include
Agrobacterium tumerfacium, which uses a pilus-like structure which
binds to the plant cell via specific receptors, and then through a
process that resembles bacterial conjugation, delivers at least
some of its content to the plant cell.
[0127] Set forth below are examples of cell lines to which RNA can
be delivered according to the method of this invention.
[0128] Examples of human cell lines include but are not limited to
ATCC Nos. CCL 62, CCL 159, HTB 151, HTB 22, CCL 2, CRL 1634, CRL
8155, HTB 61, and HTB104.
[0129] Examples of bovine cell lines include ATCC Nos. CRL 6021,
CRL 1733, CRL 6033, CRL 6023, CCL 44 and CRL 1390.
[0130] Examples of ovine cells lines include ATCC Nos. CRL 6540,
CRL 6538, CRL 6548 and CRL 6546.
[0131] Examples of porcine cell lines include ATCC Nos. CL 184, CRL
6492, and CRL 1746.
[0132] Examples of feline cell lines include CRL 6077, CRL 6113,
CRL 6140, CRL 6164, CCL 94, CCL 150, CRL 6075 and CRL 6123.
[0133] Examples of buffalo cell lines include CCL 40 and CRL
6072.
[0134] Examples of canine cells include ATCC Nos. CRL 6213, CCL 34,
CRL 6202, CRL 6225, CRL 6215, CRL 6203 and CRL 6575.
[0135] Examples of goat derived cell lines include ATCC No. CCL 73
and ATCC No. CRL 6270.
[0136] Examples of horse derived cell lines include ATCC Nos. CCL
57 and CRL 6583.
[0137] Examples of deer cell lines include ATCC Nos. CRL
6193-6196.
[0138] Examples of primate derived cell lines include those from
chimpanzee's such as ATCC Nos. CRL 6312, CRL 6304, and CRL 1868;
monkey cell lines such as ATCC Nos. CRL 1576, CCL 26, and CCL 161;
orangautan cell line ATCC No. CRL 1850; and gorilla cell line ATCC
No. CRL 1854.
[0139] 4. Pharmaceutical Compositions
[0140] In a preferred embodiment of the invention, the invasive
bacteria containing the RNA molecules, and/or DNA encoding such,
are introduced into an animal by intravenous, intramuscular,
intradermal, intraperitoneally, peroral, intranasal, intraocular,
intrarectal, intravaginal, intraosseous, oral, immersion and
intraurethral inoculation routes.
[0141] The amount of the live invasive bacteria of the present
invention to be administered to a subject will vary depending on
the species of the subject, as well as the disease or condition
that is being treated. Generally, the dosage employed will be about
10.sup.3 to 10.sup.11 viable organisms, preferably about 10.sup.5
to 10.sup.9 viable organisms per subject.
[0142] The invasive bacteria of the present invention are generally
administered along with a pharmaceutically acceptable carrier
and/or diluent. The particular pharmaceutically acceptable carrier
an/or diluent employed is not critical to the present invention.
Examples of diluents include a phosphate buffered saline, buffer
for buffering against gastric acid in the stomach, such as citrate
buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0)
alone (Levine et al. J. Clin. Invest., 79:888-902 (1987); and Black
et al J. Infect. Dis., 155:1260-1265 (1987)), or bicarbonate buffer
(pH 7.0) containing ascorbic acid, lactose, and optionally
aspartame (Levine et al. Lancet, 11:467-470 (1988)). Examples of
carriers include proteins, e.g., as found in skim milk, sugars,
e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers
would be used at a concentration of about 0.1-30% (w/v) but
preferably at a range of 1-10% (w/v).
[0143] Set forth below are other pharmaceutically acceptable
carriers or diluents which may be used for delivery specific
routes. Any such carrier or diluent can be used for administration
of the bacteria of the invention, so long as the bacteria are still
capable of invading a target cell. In vitro or in vivo tests for
invasiveness can be performed to determine appropriate diluents and
carriers. The compositions of the invention can be formulated for a
variety of types of administration, including systemic and topical
or localized administration. Lyophilized forms are also included,
so long as the bacteria are invasive upon contact with a target
cell or upon administration to the subject. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
composition, e.g., bacteria, of the invention can be formulated in
liquid solutions, preferably in physiologically compatible buffers
such as Hank's solution or Ringer's solution.
[0144] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0145] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner.
[0146] For administration by inhalation, the pharmaceutical
compositions for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebuliser, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the composition, e.g.,
bacteria, and a suitable powder base such as lactose or starch.
[0147] The pharmaceutical compositions may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0148] The pharmaceutical compositions may also be formulated in
rectal, intravaginal or intraurethral compositions such as
suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
[0149] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the bacteria of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art,
so long as the bacteria are still invasive upon contact with a
target cell.
[0150] The compositions may, if desired, be presented in a pack or
dispenser device and/or a kit which may contain one or more unit
dosage forms containing the active ingredient. The pack may for
example comprise metal or plastic foil, such as a blister pack. The
pack or dispenser device may be accompanied by instructions for
administration.
[0151] The invasive bacteria containing the RNA or RNA-encoding DNA
to be introduced can be used to infect animal cells that are
cultured in vitro, such as cells obtained from a subject. These in
vitro-infected cells can then be introduced into animals, e.g., the
subject from which the cells were obtained initially,
intravenously, intramuscularly, intradermally, or
intraperitoneally, or by any inoculation route that allows the
cells to enter the host tissue. When delivering RNA to individual
cells, the dosage of viable organisms to administered will be at a
multiplicity of infection ranging from about 0.1 to 10.sup.6,
preferably about 10.sup.2 to 10.sup.4 bacteria per cell.
[0152] In yet another embodiment of the present invention, bacteria
can also deliver RNA molecules encoding proteins to cells, e.g.,
animal cells, from which the proteins can later be harvested or
purified. For example, a protein can be produced in a tissue
culture cell.
[0153] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0154] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references including literature
references, issued patents, published patent applications as cited
throughout this application are hereby expressly incorporated by
reference. The practice of the present invention will employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene
Transfer
[0155] Vectors For Mammalian Cells (J. H. Miller and M. P. Calos
eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,
Vols. 154 and 155 (Wu et al. eds), Immunochemical Methods In Cell
And Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987); Handbook Of Experimental Immunology, Volumes I-IV
(D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986).
EXAMPLES
Methods
[0156] siRNA-Generating Plasmid Construction:
[0157] Oligonucleotides were obtained at 0.2 .mu.mol from QIAGEN
with PAGE purification. After annealing, oligonucleotides were
inserted into the BamHI and HindIII binding sites within pSilencer
2.0-U6 (Ambion, Inc.) according to the manufacturer's
instructions.
[0158] The following sequences were used:
[0159] The k-Ras-1 and -2 64-mers target the nucleotides encoding
for amino acids 9-15 of k-Ras protein which spans the specific
mutation of V12.
TABLE-US-00001 k-Ras-1 (64-mer): (SEQ ID NO: 1)
5'gATCCCgTTggAgCTgTTggCgTAgTTCAAgAgACTACgCCAACAgCTCCAACTTTTTTggAAA3'
k-Ras-2 (64-mer): (SEQ ID NO: 2)
5'AgCTTTTCCAAAAAAgTTggAgCTgTTggCgTAgTCTCTTgAACTACgCCAACAgCTCCAACgg3'
[0160] The .beta.-Catenin-1 and -2 64-mers target the nucleotides
encoding for amino acids 79-85 within the catenin protein
TABLE-US-00002 .beta.-Catenin-1 (64 mer): (SEQ ID NO: 3)
5'gATCCCAgCTgATATTgATggACAgTTCAAgAgACTgTCCATCAATA
TCAgCTTTTTTTggAAA3' .beta.-Catenin-2 (64 mer): (SEQ ID NO: 4)
5'AgCTTTTCCAAAAAAAgCTgATATTgATggACAgTCTCTTgAACTgT
CCATCAATATCAgCTgg3'
[0161] The EGFP-1 and -2 64-mers target the nucleotides encoding
for amino acids 22-28 of EGFP.
TABLE-US-00003 EGFP-1 (64-mer): (SEQ ID NO: 5)
5'gATCCCgACgTAAACggCCACAAgTTTCAAgAgAACTTgTggCCgTT
TACgTCTTTTTTggAAA3' EGFP-2 (64-mer): (SEQ ID NO: 6)
5'AgCTTTTCCAAAAAAgACgTAAACggCCACAAgTTCTCTTgAAACTT
gTggCCgTTTACgTCgg3'
Transkingdom RNA Interference Plasmid Construction:
[0162] The engineered plasmid pT7RNAi-Hly-Inv, TRIP was constructed
from pGB2.OMEGA.inv-hly (Milligan et al., Nucleic Acids Res. 15,
8783 (1987)) and pBlueScript II KS(+). Oligonucleotides containing
multiple cloning site (MCS), T7 promoter, enhancer and terminator
(synthesized from Qiagen) were ligated into blunted BssHII sites of
KSII(+), and .beta.-catenin hairpin oligos were inserted into BamHI
and SalI sites of MCS to generate plasmid pT7RNAi. PstI fragments
containing the inv locus of pGB2.OMEGA.inv-hly were inserted into
PstI site of KSII(+). Using pGB2.OMEGA.inv-hly as template, H1yA
gene was amplified by PCR (Pfx DNA polymerase, Invitrogen Inc.)
with primers, hly-1: 5'-CCCTCCTTTGATTAGTATATTCCTATCTTA-3' (SEQ ID
NO:7) and hly-2: 5'-AAGCTTTTAAATCAGCAGGGGTCTTTTTGG-3' (SEQ ID
NO:8), and were cloned into EcoRV site of KSII(+)/Inv. Hly-Inv
fragment was excised with BamHI and SalI. After blunting, it was
ligated into EcoRV site incorporated within T7 terminator of
pT7RNAi
Bacteria:
[0163] The auxotrophic Salmonella typhimurium aroA 7207 (S.
typhimurium 2337-65 derivative hisG46, DEL407[aroA544::Tn10(Tc-s)]
used as the plasmid carrier in this study was kindly provided by
Prof. BAD Stocker, Stanford University, CA. Escherichia coli XL-1
Blue was used to maintain the plasmids (Strategene).
[0164] Transformation of SL 7207 was achieved using an adapted
electroporation protocol (1). Competent SL7207 and 1 .mu.g plasmid
were incubated on ice in a chilled 0.2 cm electroporation cuvette
for 5 min. A 2.5 kV, 25 .mu.F, 200.OMEGA. impulse was applied using
a BioRad Genepulser. 1 mL of prewarmed SOC medium was added and
bacteria were allowed to recover for 1 hr at 37.degree. C. with 225
RPM shaking before plating on selective agar plates. Presence of
the plasmids was confirmed using minipreparation after alcalinic
lysis and separation on 0.7% agarose gel.
[0165] For in vitro experiments, SL 7202 were grown overnight at
37.degree. C. in Luria Broth (LB) supplemented with 100 .mu.g/mL
Ampicillin (for SL-siRAS, SL-siGFP and SL-siCAT) without shaking.
The next morning, bacteria were grown in fresh medium after 1%
inoculation from the overnight culture until reaching an OD.sub.600
of 0.4-0.6. Bacteria were centrifuged (3500 RPM, 4.degree. C.)
washed once in phosphate-buffered saline (PBS) and resuspended in
PBS at the desired concentrations. For all determinations of
bacterial number and concentration, the bacterial density was
measured spectrometrically and calculated according to the formula
c=OD.sub.600*8.times.10.sup.8/mL.
[0166] For animal experiments, SL 7207 were grown in Brain Heart
Infusion Broth (Sigma) in a stable culture overnight supplemented
with the appropriate antibiotics where required. Bacteria were
washed and resuspended in PBS at a concentration of
2.5.times.10.sup.10/mL. Serial dilutions were done and plated on
selective agar at several times during the experiment to verify the
actual number of bacteria administered.
[0167] Plasmids were also transformed into BL21DE3 strain (Gene
Therapy Systems) according to the manufacturer instructions.
Bacteria were grown at 37.degree. C. in Brain-Heart-Infusion-broth
with addition of 100 .mu.g/ml Ampicillin. Bacteria numbers were
calculated using OD.sub.600 measurement. For cell infection,
overnight cultures were inoculated into fresh medium for another 2
h growth.
Cell Culture:
[0168] A human colon cancer cell line (SW 480) was used herein. It
carries a mutation of APC protein resulting in increased basal
levels of .beta.-catenin. A stably GFP-expressing cell line derived
from yolk sac epithelium, CRL 2583 (ATCC, Rockville, Md.) was used
for GFP-knockdown experiments. CRL 2583 was maintained in 200
.mu.g/mL G418 until 30 min before bacterial infection. SW 480 were
grown in RPMI-1640 supplemented with 10% fetal bovine serum. CRL
2583 were grown in high glucose, high NaHCO.sub.3 DMEM supplemented
with 15% FBS as recommended by the supplier. All growth media were
routinely supplemented with antibiotics: 100 U/ml penicillin G, 10
.mu.g/ml streptomycin, 2.5 .mu.g/ml amphotericin (all media and
additives purchased from Sigma, St. Louis).
[0169] For direct transfection of plasmids, 500,000 cells were
seeded into 6 cm petri dishes and allowed to grow overnight before
they were transfected using a standard CaP-coprecipitation
protocol.
[0170] Briefly, 15 .mu.g plasmid-DNA are mixed in 500 .mu.L
reaction mix (2.times.HEPES buffer, 60 .mu.L CaP) and dropped to
the cells in fresh medium without FBS. Precipitation was allowed to
continue for 9 hrs before precipitates were washed away. Cells were
harvested at different time points (36, 48, 60, 72, and 96
hrs).
[0171] For standard bacterial infection assays, 500,000 cells were
seeded into 6 cm petri dishes and were allowed to attach overnight.
30 min prior to addition of the bacteria, the growth medium was
replaced with fresh medium without antibiotics and fetal bovine
serum. SL 7207-siRAS, -siCAT, -siGFP were added in 500 .mu.L PBS to
reach the designated multiplicity of infection (MOI) of 100, 500 or
1000 and infection was carried out in a standard incubator with
37.degree. C., 5% CO.sub.2. By the end of the indicated infection
period, plates were washed once with 4 ml of serum-free RPMI medium
and 3 times with 4 ml PBS, then 5 ml of fresh complete RPMI medium
containing 100 .mu.g/mL of ampicillin and 150 .mu.g/mL of
gentamycin were added. Twenty-four hours later, tetracycline was
added to final concentration of 15 .mu.g/mL. At indicated different
time points (24-96 h) after bacterial invasion, cells were
harvested for western blot or flow cytometry.
[0172] For staining of intracellular bacteria, cells were grown on
Lab-Tek II Chamber Slides (Nalgene Nunc, USA). After bacterial
invasion as described above, cells were washed with PBS and fixed
in 1% paraformaldeyde for 10 min. Acridin Orange (Sigma) solution
(0.01%) was added for 45 sec, then washed with PBS. Crystal Violet
stain (Sigma) was applied for 45 sec, then washed with PBS.
Coverslips were mounted using PERMOUNT.TM. mounting medium and
invasion was assessed using confocal microscopy.
MTT Assay:
[0173] After treatment with SL7207-siRAS and/or SL7207-siCAT, cells
were trypsinized (24 h or 48 h later), diluted and seeded into
96-well plate at a concentration of 5000 cells/well. Cells were
then allowed to grow for up to 4 days. At the desired incubation
time point, medium was removed and 100 .mu.l of MTT solution (5
mg/mL) was added to each well. After an incubation period of 4 h,
MTT solution was drained away and cells were lysed by adding 100
.mu.L of solubilization reagent (Isopropanol:1N HCl:10% SDS 43:2:5)
to each well. The resulting signal of the dark blue
formazan-product was photometrically determined at 570 nm
wavelength. The amount of color formation is dependent on the
number of surviving cells per well.
Colony Formation Test:
[0174] After treatment with SL7207-siRAS and/or SL7207-siCAT, cells
were trypsinized (24 h post-transfection), diluted and seeded into
6-well MTP at a concentration of 750 cells/well. Cells were kept
growing for two more weeks to let them form visible colonies. Two
weeks later, medium was removed and 1 ml of Giemsa stain (7.415
g/L) were added to each well. After 10-min incubation at 37.degree.
C., Giemsa stain was drained away and cells washed with PBS. Groups
of more than eight cells were counted as positive colonies.
Western Blot:
[0175] Cells were washed with chilled PBS, scraped off and lysed in
lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM
EGTA, 1% NP-40, 1 mM DTT) containing 0.1% protease inhibitor mix
(Sigma). 20 .mu.g of protein were separated using 11% SDS-Page Gel
and transferred to a 0.4 .mu.m PVDF membrane (Schleicher and
Schuell). The membrane was blocked using 5% milk and incubated for
1 hr with specific antibodies at the indicated concentrations:
Living Colors.RTM. antibody (Clontech)-1:500, .beta.-catenin
antibody (Santa Cruz)-1:500, k-Ras antibody (Santa Cruz)-1:300 and
.beta.-actin (Santa Cruz) 1:500. Each was followed by incubation
with horseradish-peroxidase conjugated anti-rabbit or anti-goat
secondary antibodies (Santa Cruz)-1:1000-1:2000. Bands were
detected using ECL.RTM. chemoluminescence detection (Amersham).
Flow Cytometry:
[0176] For flow cytometry, cells were trypsinized for 3 min,
resuspended in fresh medium and washed in PBS. After
centrifugation, cells were fixed for 10 min in 1%
paraformaldehyde/PBS at 4.degree. C. Flow cytometry was performed
using FACScan (Becton Dickinson), data analysis was done using
CellQuest.RTM. software.
Animal Techniques:
[0177] Six to eight week old female GFP+ transgenic mice
(CgTg5Nagy) were obtained from Jackson laboratories. They were
housed in the BIDMC animal research facility with ad libitum access
to standard rodent diet and drinking water. Treatment was initiated
at ten weeks of age. For the iv treatment protocol, four doses of
10.sup.6 cfu SL-siRAS or SL-siGFP dissolved in 50 .mu.L PBS were
injected into the tail vein on alternating days. Mice were weighed
daily and monitored for signs of disease.
[0178] Mice were sacrificed one day after the final treatment at
which time tissue samples were taken for histochemistry and flow
cytometric analysis. Tissues were paraffin embedded and sectioned
in 6 .mu.m steps for histochemistry and fluorescence microscopy.
For flow cytometry, hepatocyte and splenocyte suspensions were
prepared through the use of cell strainers (Falcon). Organ
suspensions were fixed in 4% formalin and flow cytometry was
performed using FACScan (Becton Dickinson), data analysis was done
using CellQuest.RTM. software.
[0179] For the Xenograft cancer model, female BALB/c nude mice
(Charles River Laboratories) were randomized into two groups (n=6).
Three weeks before treatment, 1.times.10.sup.7 of SW480 cells were
implanted subcutaneously. Treatments were initiated when the tumors
reached about 10 mm in diameter. The treatment group was injected
through tail vein with 1.times.10.sup.8 cfu of E. coli expressing
shRNA against .beta.-catenin in PBS. The control group was
similarly treated except that the E. coli contains the TRIP vector
without shRNA insert. The treatment was carried out every 5 days
for a total of three treatments. Mice were sacrificed 5 days after
the last treatment. Tissues were frozen and fixed for analysis of
.beta.-catenin mRNA level by real-time PCR and .beta.-catenin
protein level by immunohistochemistry.
[0180] For in vivo silencing experiments, female C57/BL6 mice
(Charles River Laboratories) were randomly divided into two groups.
The treatment group was administered orally with 5.times.10.sup.10
cfu E. coli expressing shRNA against .beta.-catenin in 200 .mu.L
phosphate-buffered saline (PBS). The control group was similarly
treated except that the E. coli contains the TRIP vector without
the shRNA insert. Two independent experiments were performed with 6
and 5 mice per group used, respectively. The treatment was carried
out daily for 5 days per week for a total of 4 weeks. Mice were
sacrificed 2 days after the last treatment, and tissues were
paraffin-embedded.
Immunohistochemistry
[0181] Immunostaining was performed on 6 .mu.m tissue sections
using Vectastain Elite ABC avidin-biotin staining kit (Vector
laboratories, Burlingame, Calif.) according to the instructions by
the manufacturer. Slides were deparaffinized and rehydrated using a
standard protocol. For antigen retrieval, slides were heated by
microwave in 5% urea for 5 min. Unspecific binding sites were
blocked with 1% bovine serum albumin for 10 min and endogenous
peroxidase activity was suppressed by treatment with 3%
H.sub.2O.sub.2 in methanol for 10 min. Sections were exposed to
primary antibody LIVING COLORS.TM. rabbit polyclonal antibody
(Clontech) at 1:500 dilution overnight at 4.degree. C. The
chromogen used was 3,3'Diamino-enzidine (DAB) (Vector),
counterstaining was done with hematoxyline.
Interferon Response Detection:
[0182] SW480 cells were treated for 2 h with E. coli transformed
with the TRIP encoding shRNA against human .beta.-catenin or mutant
k-Ras at MOI of 1:1000. Untreated cells were used as control. Cells
were harvested at 24, 48 and 72 h. The expression levels of OAS1,
OAS2, MX1, ISGF3.gamma. and IFITM1 genes were determined by RT-PCR
using the Interferon Response Detection Kit (SBI System
Biosciences, CA).
Example 1
Knock Down of Green Fluorescent Protein Using Bacteria Mediated
Gene Silencing In Vitro and In Vivo
[0183] In the following experiments, an attenuated strain of
Salmonella typhimurium (SL 7207, obtained from BAD Stocker,
Stanford University) was used. To prove that the concept is useful
as a general approach, we did confirmation experiments also with
another attenuated strain of Salmonella typhimurium (VNP 70009,
obtained from VION Pharmaceuticals, New Haven) and an invasive and
attenuated strain of E. coli (BM 2710, obtained from P. Courvalin,
Institut Pasteur, Paris).
[0184] Silencing plasmids were designed based on a commercially
available plasmid (pSilencer, Ambion) to knock down the target
genes GFP, .beta.-catenin and oncogenic k-Ras (V12G). These
plasmids were transformed into SL 7207 by electroporation and
positive clones were verified by growth on selective agar and DNA
preparation.
[0185] For in vitro use, knockdown of GFP expression was
demonstrated using the stable GFP+ cell line CRL 2598 (ATCC,
Rockland, Va.). Knockdown of oncogenic k-Ras (V12G) and
.beta.-catenin was demonstrated using the colon cancer cell line SW
480 and the pancreatic cancer cell line CAPAN-1.
[0186] A system of bacterial delivery using an invasive bacterial
strain, S. typhimurium, was developed with a commercially available
eukaryotic transcription plasmid, pSilencer (Ambion). The S.
typhimurium strain SL 7207 (kindly provided by B. Stocker, Stanford
University) is attenuated through an auxotrophy in the synthesis
pathway for aromatic amino acids, and dies quickly after invasion
into a target cell due to lack of nutrients. This strain has been
used successfully for delivery of DNA in vitro and in mouse models,
mainly with the purpose of DNA vaccination.
[0187] To verify bacterial entry into epithelial cells, an invasion
assay was performed. SW 480 cells were infected for 2 hrs with
SL-siRAS followed by 2 hrs of treatment with gentamycin. Acridin
orange/crystal violet staining revealed good invasion efficiency.
90% of the SW 480 cells harbored viable SL 7207 bacteria. The
average number of intracellular bacteria was 6 (range, 2-8) (FIG.
1). (Micrograph A1 is the transmission image. Micrograph A2 is the
fluorescent image. Micrograph A3 is the merged image.) The number
of viable intracellular bacteria reduced quickly over time. After
24 hrs and 48 hrs, only 10% and 3% of cells were found to still
contain bacteria.
[0188] In the next experiment, The effective reduction of GFP
expression in the GFP+ cell line was demonstrated. Successful
knockdown of oncogenes k-Ras and .beta.-catenin was confirmed using
Western blot and RT-PCR. Oncogene knockdown resulted in growth
retardation and decreased tumor formation in a xenograft animal
model.
[0189] Cells stably expressing GFP(CRL 2583) were infected with
SL7207 carrying pSilencer2.0 including a sequence to silence GFP
mRNA (SL-siGFP). (See above). After 48 hrs, cells treated with
SL-siGFP showed a marked decrease in GFP expression as compared to
cells treated with SL-siRAS and untreated control (FIGS. 1B and
1C). Treatment with SL-siGFP led to loss of GFP signal in a manner
dependent on the multiplicity of infection (MOI) applied. In
untreated cells, only 4.3% display low or absent fluorescence. In
SL-siGFP treated cells, this fraction increased to 78.1% (treated
with MOI 1:500) and 92.3% (MOI 1:1000). Control treatment with
SL-siRAS lead to a slight loss of fluorescence (7.5% at MOI 1:500
and 8.4% at MOI 1:1000). This is also shown in the fluorescence
microscope photograph in FIG. 1C. The top micrograph is
(200.times.) of SL-siRAS and the bottom of SL-siGFP (below) treated
CRL 2583 cells. This finding was confirmed using flow
cytometry.
[0190] In a series of animal experiments with stably GFP-expressing
mice, we were able to demonstrate knockdown of GFP expression in
the liver and in the colon (in both organs approx 50% reduction of
GFP expression) after oral and intravenous application of SL 7207
carrying the GFP-silencing plasmid.
[0191] In animal experiments, S. typhimurium was used to achieve
gene silencing in a transgenic mouse model (GFP+). Using this
method, silencing of the transgene in the animal experiment is
demonstrated at mRNA level as well as on protein levels and tissue
sections of various organs (liver, gastrointestinal tract) with
limited toxicity.
Example 2
Knock Down of k-Ras and .beta.-Catenin Using Bacteria Mediated Gene
Silencing
[0192] Next, BMGS was applied to knock down a specific
disease-related gene. The specific oncogenic point mutation in the
k-Ras gene, k-Ras.sup.V12G, which is present in the human colon
carcinoma cell line, SW 480 was targeted.
[0193] After construction of the silencing plasmids and before they
were electroporated into the attenuated SL7207, their activity was
tested by transfecting them into SW 480 cells using CaP
coprecipitation.
[0194] Western blot (FIG. 4A) shows efficient knockdown of k-Ras
using the pSilencer-kras (V12G) insert at 36 h and 48 h
posttransfection. At later time points, the protein expression
recovers, which is due to outgrowth of transfected clones which
have a growth disadvantage versus non transfected clones in which
the oncogenic k-Ras would still drive replication. When BMGS with
SL7207 as a carrier was used to mediate the knockdown, k-Ras levels
were decreased at MOI of 1:500 and 1:1000. Using BMGS, knockdown of
the k-Ras protein was observed with similar efficiency compared to
direct transfection of the silencer plasmid using calcium-phosphate
coprecipitation, although the onset of knockdown was slightly
delayed by 12 hrs. (FIG. 4A). With an MOI of 1:1000, the result can
be observed for a longer time (up to 72 hrs) (FIG. 2A).
[0195] The Western blot for .beta.-catenin (FIG. 4B) shows delayed
knockdown after transfection, with a maximum effect seen at 96 hrs
post transfection. It is assumed that this delay is caused by the
survival time of SL 7207 intracellularly before the plasmid is
liberated (FIG. 2A).
[0196] After treatment with SL-siRAS and resulting knockdown of the
oncogenic k-Ras (V12G), SW 480 cells displayed significantly
reduced viability and colony formation ability (FIG. 2B). Cells
were coincubated with equal amounts (2.5.times.10.sup.8) of
SL-siRAS and SL-siCAT bacteria. Control cells were treated with
untransformed SL 207. 48 hrs after transfection, cells were seeded
in 96 well plates for MTT test and 6 well plates for colony
formation assays. At 120 hrs after treatment, viability, as
assessed by MTT assay, was reduced to 62.5% after SL-siRAS
treatment and 51% after SL-siCAT treatment. Combined treatment
further reduced viability to 29% of control treated cells. SL-siRAS
treatment and SL-siCAT treatment reduced the ability of SW 480 to
form colonies by 37.7% and 50%, respectively. Combined treatment
lead to 63.3% reduction.
[0197] Further, treatment with SL-RAS completely inhibited the
tumor formation ability of SW 480 cells when injected
subcutaneously into nude mice, while treatment with empty SL 7207
did not influence their ability to form tumors (FIG. 2C). SW 480
cells (untreated, treated with SL 7207 or SL-siRAS) were
subcutaneously injected into nude mice (4.times.10.sup.6 cells, n=4
animals per group). Pretreatment with SL-siRAS completely abolished
the ability to form tumors (no tumors visible in any of the four
animals, day 40) (FIG. 2C).
[0198] To test whether this approach can be employed universally,
another cancer-related gene, .beta.-catenin was targeted (FIG. 2A).
Basal levels of .beta.-catenin are high in SW 480 cells, due to a
mutated APC-gene, but can be reduced through treatment with hairpin
siRNA after pSilencer(siCAT) transfection (FIG. 2A). After
treatment with SL 7207 carrying pSilencer with the .beta.-catenin
construct (SL-siCAT), significant knockdown of .beta.-catenin
expression was achieved which resulted in decreased viability and
colony formation ability (FIG. 2A). .beta.-catenin was knocked down
from 96 hr, but recovered from 144 hr.
Example 3
In Vivo Bacterial Mediated Gene Silencing
[0199] The method of bacterial mediation of RNAi offers the
possibility of selectively targeting more than one gene at a time
which might allow for increased efficiency for future applications,
e.g. anticancer treatment through interference with multiple
oncogenic pathways. To test the feasibility of such an approach,
both the mutated k-Ras oncogene and .beta.-catenin were targeted
simultaneously. After simultaneous treatment with SL-siRAS and
SL-siCAT, knockdown of both genes was observed at the protein level
and resulted in further decreased viability and colony formation
ability (FIG. 2). These findings demonstrate that the proposed
concept of bacterial mediated gene silencing can be successfully
used in vitro for different target genes and in different cell
lines.
[0200] A mouse model was chosen to test whether this approach can
be used to silence target genes in vivo. CgTg5-Nagy mice express
high levels of GFP in all tissues. 14 mice were randomly assigned
to receive four doses of 10.sup.6 cfu of either SL-siGFP or
SL-siRAS i.v. on alternating days (seven animals per group). This
treatment was well tolerated with no weight loss or clinically
apparent signs of disease. All mice were sacrificed one day after
the last treatment.
[0201] Liver tissue slides were assessed by fluorescence microscopy
and immunohistochemistry with GFP antibody. (FIG. 3A). Intravenous
treatment with SL-siGFP led to decrease of fluorescence in the
liver sections of the treated animals compared with SL-siRAS
treated control animals. Histochemistry staining, with anti-GFP
antibody, verified that changes in fluorescence were caused by a
reduction in GFP and not caused by changes in background
fluorescence levels. (FIG. 5) To verify that reductions in
fluorescence in the liver sections of treated mice are really
caused by changes in GFP expression levels and not due to changes
in background fluorescence, tissue slides were stained with GFP
specific antibody.
[0202] Immunohistochemical staining patterns correlate well with
fluorescence microscopy images and confirm that changes in
fluorescence are caused by decreased GFP expression. Fluorescence
microscopy (50.times.) and corresponding immunohistochemistry image
(50.times.) of liver section from control (top row) and iv treated
(lower row) animal.
[0203] Staining patterns correlated well with fluorescence images.
Subsequent image analysis revealed reductions in the number of GFP
expressing cells between 9-25% after SL-siGFP treatment. These
findings were confirmed by flow cytometric analysis of single cell
suspensions of hepatocytes which showed a significant decrease in
the number of GFP-positive hepatocytes in SL-siGFP treated vs
SL-siRAS treated animals (FIG. 3B). Flow cytometry measurements of
hepatocyte and splenocyte suspensions were performed. After
intravenous treatment with SL-siGFP, the number of GFP+ hepatocytes
was significantly reduced compared to control treated (SL-siRAS)
animals (SL-siRAS: 50.0% [45.4-53.2%], SL-siGFP: 39.9%
[26.1-53.2%], p<0.05).
[0204] These results indicate that significant gene silencing can
be achieved in vivo using this approach. Using iv application of
attenuated S. typhimurium we were able to extend the in vitro
findings into a mouse model and achieve significant gene silencing
in the liver. Other organs might become accessible through use of
different invasive bacterial strains or different routes of
application. Especially professional phagocytes will be a promising
target for bacteria-mediated gene silencing, as transfection
efficiencies have been reported to be higher for these cells
compared to cells of epithelial lineage.
Example 4
Transkingdom RNA Interference
[0205] The use of bacteria-mediated RNAi in higher organisms holds
the potential for functional genomics in mammalian system, as
previously demonstrated in C. elegans, and for other in vivo
applications of RNAi. To investigate this possibility, the
bacterial plasmid pT7RNAi-Hly-Inv, termed TRIP (transkingdom RNA
interference plasmid) was constructed (FIG. 6A). In this novel
plasmid construct, the expression of shRNA was directed under the
bacteriophage T7 promoter (Milligan and Uhlenbeck, Methods Enzymol.
180, 51-62 (1989) and Milligan et al., Nucleic Acids Res. 15,
8783-8798 (1987), rather than a mammalian promoter or enhancer. The
shRNA can only be produced by the bacterial system. The TRIP vector
contains the Inv locus that encodes invasion (Isberg et al., Cell
50, 769-778 (1987)), which permits the non-invasive E. coli to
enter .beta.1-integrin-positive mammalian cells (Young et al., J.
Cell Biol. 116, 197-207 (1992)). The TRIP vector also contains the
Hly A gene that encodes listeriolysin O to permit genetic materials
to escape from entry vesicles (Mathew et al., Gene Ther. 10,
1105-1115 (2003) and Grillot-Courvalin et al., Nat. Biotechnol. 16,
862-866 (1998)). TRIP constructs were introduced into a competent
strain of non-pathogenic E. coli, BL21DE3, which contains T7 RNA
polymerase to express shRNA. A TRIP against the cancer gene
.beta.-catenin was constructed as an example. Activation of the
.beta.-catenin pathway from over-expression or oncogenic mutation
of .beta.-catenin is responsible for the initiation of the vast
majority of colon cancers and is involved in a variety of other
cancer types (Kim et al., Oncogene 24, 597-604 (2005)). Despite the
potential of .beta.-catenin as a cancer therapeutic target, the
.beta.-catenin pathway has been recalcitrant to inhibition by small
molecules. .beta.-catenin is a preferred choice in proof of concept
experiments for testing the potency of new a RNAi approach because
it is commonly stabilized in cancer cells. TRIP can be modified to
enable bacteria to express interfering RNA against various genes of
interest.
[0206] To determine if gene silencing can be achieved through this
transkingdom system, human colon cancer cells (SW 480) were
co-cultured in vitro with E. coli for 2 h (FIGS. 6B and 6D) or
different time (FIG. 6C), then treated with antibiotics to remove
extracellular bacteria. Cells were further cultured for 48 h before
harvest for analysis of gene silencing. As shown in FIG. 6B-6D,
.beta.-catenin was potently and specifically silenced at protein
and mRNA level, while .beta.-actin, k-Ras, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were not affected.
To further test the specificity of the transkingdom RNAi, E. coli
containing the TRIP against mutant k-Ras (GGT.fwdarw.GTT at codon
12) silenced k-Ras expression in SW 480 cells with the same codon
12 mutation, but not in DLD1 cells with mutation in a different
codon of k-Ras (GGC.fwdarw.GAC at codon 13, FIG. 6E). As an shRNA
control, E. coli containing the TRIP against wild type k-Ras
exerted no gene-silencing effect on mutated k-Ras in SW 480 cells
(FIG. 6F). These results show that the transkingdom RNA
interference is highly gene-specific, sufficient to discriminate a
point mutation.
[0207] To investigate the variables that affect the potency of gene
silencing by the transkingdom system, cells were incubated for 2 h
with the E. coli at different multiplicity of infection (MOD. As
shown in FIG. 6B, the potency of gene silencing was dependent on
MOI, with near complete gene silencing at a MOI of 1:1,000. To
determine the effect of co-culture time on gene silencing, cells
were incubated with the E. coli at a MOI of 1:500 for different
times. As shown in FIG. 6C, gene-silencing potency increased with
incubation time up to 2 h. The dependency of gene silencing on MOI
and co-culture time provides controllable flexibility for gene
silencing in various applications.
[0208] To further confirm that the .beta.-catenin gene silencing is
mediated specifically by shRNA, identification of the specific
cleavage fragment of .beta.-catenin mRNA was attempted by using
5'-RACE (rapid amplification of cDNA ends) PCR technique. A
specific hallmark of RNAi-mediated gene silencing is the cleavage
of .beta.-catenin mRNA at the specific sites of the mRNA as
predicted from the shRNA sequence. Based on the time course of
.beta.-catenin silencing (FIG. 7A), total RNA was isolated from SW
480 cells 8 h and 16 h after treatment with E. coli expressing
shRNA against .beta.-catenin to identify the cleaved fragments of
mRNA. The cleaved .beta.-catenin mRNA was found as early as 8 h
after treatment with E. coli expressing shRNA: no fragments were
detected in the control (FIGS. 7B and 7C). The sequence analysis of
the cleaved intermediate of .beta.-catenin mRNA confirms the
cleavage site located within the targeting sequence. This result
shows that shRNA produced by bacteria trigger specific cleavage of
the .beta.-catenin mRNA through the RNAi-mediated gene
silencing.
[0209] Induction of interferon response has been reported as a
potential challenge to the specificity of some RNAi approaches
(Bridge et al., Nat. Genet. 34, 263-264 (2003) and Hornung et al.,
Nat. Med. 11, 263-270 (2005)). To test if the gene silencing
induced by the transkingdom RNAi is associated with interferon
response induction, key interferon response genes were measured.
The 2',5'-oligoadenlylate synthetases (OAS1 and OAS2) are important
interferon-induced genes for the inhibition of cellular protein
synthesis after viral infection. MX1 gene, a member of the
interferon-induced myxovirus resistance protein family (MX
proteins), participates in the innate host defense against RNA
viruses. IFITM1, a member of the interferon-inducible transmembrane
proteins, mediates the anti-proliferation activity of interferon.
ISGF3.gamma. is part of a cellular interferon receptor involved in
interferon-induced transcription regulation and stimulation. These
genes have been used as a standard panel for analyzing interferon
response induction by interfering RNA (Interferon Response
Detection Kit, SBI Systems Biosciences, CA). The mRNA of the
five-interferon response genes were analyzed with semiquantitative
RT-PCR. As shown in FIG. 7D, no induction of OAS1, OAS2, MX1,
ISGF3.gamma. and IFITM1 was detected following treatment with E.
coli encoding shRNA against .beta.-catenin. These data show that
gene silencing induced by transkingdom RNAi is not associated with
non-specific interferon response induction.
[0210] The mechanism of the transkingdom RNAi transfer was
investigated. To determine if cellular entry of E. coli is required
to induce RNAi, the gene-silencing activity of E. coli was compared
with or without the Inv locus. The Inv encodes invasin that
interacts with .beta.1-integrin to facilitate the entry of E. coli
into the cells. As expected, E. coli without Inv failed to enter
cells (FIG. 8A). Surprisingly, Inv alone is not sufficient to
enable E. coli to enter colon cancer cells (FIG. 8A), and no
detectable gene silencing was observed in the absence of
intracellular bacteria (FIG. 8B). The Hly A gene was introduced,
which is thought to facilitate delivered genetic materials to
escape from the entry vesicles (Grillot-Courvalin et al., Nat.
Biotechnol. 16, 862-866 (1998). As expected, Hly alone failed to
enable cell entrance of E. coli, but commensal E. coli with both
Inv and Hly entered colon cancer cells with high efficiency (FIG.
8A). Under these conditions .beta.-catenin was potently silenced up
to 96 h (FIG. 8C). These results show that E. coli require both Inv
and Hly to enter cells to induce transkingdom RNAi.
[0211] To determine whether gene silencing requires continued
bacterial replication inside target cells, tetracycline was
employed to block intracellular bacterial replication and
gentamycin to remove extracellular bacteria. SW 480 cells were
incubated with E. coli for 2 h followed by tetracycline treatment
initiated at different times. As shown in FIG. 8D, following the
initial 2 h infection time, an additional 2 h incubation time
without tetracycline induced near maximum gene silencing; further
delay in tetracycline treatment had no further enhancing effect on
the degree of gene silencing. Surprisingly, there was no evidence
of significant intracellular bacterial replication in the absence
of tetracycline at 6 h and 48 h (FIG. 8E), which is likely due to
the function of lysosomes and other intracellular anti-bacterial
mechanisms (Roy et al., Science 304, 1515-1518 (2004) and
Battistoni et al., Infect. Immun. 68, 30-37 (2000). These results
show that transkingdom RNAi is not dependent on persistent
bacterial replication inside target cells after the initial
infection (2 h) and incubation time (2 h).
[0212] It was next determined if the transkingdom RNAi approach
works in vivo. E. coli expressing shRNA against .beta.-catenin were
administered to mice orally. An inoculum of 5.times.10.sup.10 was
administered orally five times per week, which is comparable to a
human dosage of the probiotic E. coli Nissle 1917. Most of the
inoculum is eliminated during passage through the bactericidal
environment in the upper GI tract. Mice were treated with E. coli
expressing shRNA against mouse .beta.-catenin or with E. coli
containing the corresponding plasmid vector. Treatment was
continued for four weeks before the analysis of gene silencing by
immunohistochemistry. As shown in FIGS. 9A and 9B, .beta.-catenin
expression was silenced in the intestinal epithelium by E. coli
expressing .beta.-catenin shRNA (P<0.01), not by the control E.
coli. As a control, GAPDH expression was not reduced (FIG. 10). The
gene silencing effect was more pronounced in the regions of or
adjacent to the Peyer's patches (FIG. 9B). Treatment was well
tolerated with no gross or microscopic signs of epithelial damage
or ulcerations (FIG. 9B). These results show that mammals respond
to E. coli expressing specific shRNA with powerful local RNAi in
vivo.
[0213] The transkingdom RNAi approach was investigated to determine
if it can be used to silence a disease gene after systemic dosing.
Intravenous administration of therapeutic bacteria has been tested
in clinical trials with demonstrated safety in cancer patients
(Toso et al., J. Clin. Oncol. 20, 142-52 (2002)). Nude mice with
xenografted human colon cancer were treated intravenously with of
10.sup.8 cfu of E. coli encoding shRNA against human
.beta.-catenin. Three doses were given at a 5-day interval. The
treatments were well tolerated without adverse effects. As shown in
FIG. 9, treatment with E. coli encoding shRNA against
.beta.-catenin resulted in significant decrease in .beta.-catenin
mRNA (p<0.005, FIG. 9C) and protein (p<0.01, FIGS. 9D and 9E)
in the tumor tissues. These data show that bacteria-mediated
transkingdom RNAi can silence a disease gene in a distant part of
the body after systemic administration.
[0214] These results show that gene silencing can be achieved
through a transkingdom system. Importantly, the potency and
specificity of RNAi is preserved. Non-pathogenic E. coli has been
used clinically as probiotics with demonstrated safety (Rembacken
et al., Lancet 354, 635 (1999)). Therefore, this transkingdom
system provides a practical and clinically compatible way to
deliver RNA interference for medical indications. This E.
coli-based RNAi technology also provides a convenient vector system
for conducting RNAi-based functional studies of genes. Finally, the
results invite an intriguing possibility that such exchange of
interfering RNA may occur in nature under cohabitive, infectious,
or symbiotic conditions.
Sequence CWU 1
1
8164DNAArtificial sequenceChemically synthesized primer 1gatcccgttg
gagctgttgg cgtagttcaa gagactacgc caacagctcc aacttttttg 60gaaa
64264DNAArtificial sequenceChemically synthesized primer
2agcttttcca aaaaagttgg agctgttggc gtagtctctt gaactacgcc aacagctcca
60acgg 64364DNAArtificial sequenceChemically synthesized primer
3gatcccagct gatattgatg gacagttcaa gagactgtcc atcaatatca gctttttttg
60gaaa 64464DNAArtificial sequenceChemically synthesized primer
4agcttttcca aaaaaagctg atattgatgg acagtctctt gaactgtcca tcaatatcag
60ctgg 64564DNAArtificial sequenceChemically synthesized primer
5gatcccgacg taaacggcca caagtttcaa gagaacttgt ggccgtttac gtcttttttg
60gaaa 64664DNAArtificial sequenceChemically synthesized primer
6agcttttcca aaaaagacgt aaacggccac aagttctctt gaaacttgtg gccgtttacg
60tcgg 64730DNAArtificial sequenceChemically synthesized primer
7ccctcctttg attagtatat tcctatctta 30830DNAArtificial
sequenceChemically synthesized primer 8aagcttttaa atcagcaggg
gtctttttgg 30
* * * * *